Methamphetamine-like hapten compounds, linkers, carriers and compositions and uses thereof

ABSTRACT

The invention generally relates to hapten compounds comprising either (+) methamphetamine or (+) amphetamine conjugated to a linker. Generally speaking, hapten compounds of the invention may be used to elicit an immune response to one or more of (+) methamphetamine, (+) amphetamine, or (+) MDMA.

PRIORITY STATEMENT

This application is a continuation in part of U.S. application Ser. No.10/255,462, filed Sep. 26, 2002, which is a continuation in part of U.S.application Ser. No. 09/839,549, filed Apr. 20, 2001, now U.S. Pat. No.6,669,937, which claims the benefit of provisional U.S. Application Ser.No. 60/198,902, filed Apr. 20, 2000, now abandoned.

GOVERNMENTAL RIGHTS

This invention was made with government support under the NationalInstitute on Drug Abuse grant Nos. DA11560, DA14361, and DA05477. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention generally relates to hapten compounds comprising either(+)methamphetamine or (+)amphetamine conjugated to a linker. Generallyspeaking, hapten compounds of the invention may be used to elicit animmune response to one or more of (+) methamphetamine, (+) amphetamine,or (+) 3,4-methylenedioxymethamphetamine ((+) MDMA).

BACKGROUND OF THE INVENTION

Knowledge gained from basic research into the neurobiology of drug abusehas led to major discoveries in medicine. Nevertheless, the developmentof medical strategies for treating the complex array of neurologicalproblems associated with drug abuse has been frustratingly slow. Inparticular, development of medical treatments for alleviating theadverse psychosocial and health effects of d-methamphetamine and similarstimulants is badly needed.

d-Methamphetamine-related hospital emergency cases across the U.S.increased 256% from 1991 to 1994 (Collings, 1996). Toxic effects due toexcessive d-methamphetamine use led to more than 10,000 hospital visitseach year between 1994 and 1999 and were responsible for more than 2,000deaths over those same 5 years (Drug Abuse Warning Network, December2000). The 1995 Toxic Exposure Surveillance System data showed therewere 7,601 people treated in health care facilities for amphetamine-likedrugs and other stimulants. This is particularly striking since duringthe same period there were only 3,440 cases of cocaine treatment and atotal of 5,170 cases of all types of legal and illegal narcotics(including morphine, codeine and heroin). The current rise ind-methamphetamine use is also alarming because, unlike cocaine, it doesnot have to be imported. Even an amateur chemist can synthesize thisdrug in his home using easily obtained reagents and equipment.

Methamphetamine overdose patients can be hyperactive, agitated, andparanoid; and even one-time use of a high dose can lead to a psychoticstate lasting several days or weeks. Other complications includehyperthermia, seizures, hypertension, and cardiotoxicity. Recent studiessuggest that signs of neurotoxicity (e.g., decreased dopaminetransporter density) are present in chronic d-methamphetamine abusers,and these changes appear to correlate with a decrease in cognitivefunction.

Because there are no specific pharmacological therapies ford-methamphetamine overdose, patients receive palliative and supportivecare for symptoms while waiting for the drug to be eliminated bymetabolism and renal excretion. Emergency care of patients includesmaintenance of ventilation, hydration, electrolyte balance and controlof body temperature. Some physicians also choose to administermedications to treat seizures, agitation, or hypertensive crises. Suchtreatments can aid in managing patients' symptoms, but they do sowithout removing the causative agent. It would be advantageous to have amedication that could quickly antagonize d-methamphetamine effects byremoving the drug from the central nervous system, thereby reversingmany of the acute toxicities and reducing the potential for long-termneurological damage.

One reason why clinically effective d-methamphetamine agonists orantagonists have not been discovered is that d-methamphetamine acts atseveral sites in the central nervous system through multiple mechanismsof action. These mechanisms include, but are not limited to, disruptionof vesicular storage of dopamine, inhibition of monoamine oxidase,increased dopamine and serotonin release, and inhibition of dopamine andserotonin reuptake by their respective transporters. In addition, it islikely that any chemical antagonist (or agonist) would share at leastsome of the adverse effects of d-methamphetamine (e.g., disruption ofneurotransmitter homeostasis).

One biologically based approach to treat drug overdose is the use ofhigh-affinity, drug-specific antibodies or Fab fragments. In addition tobeing relatively safe, except for occasional allergic reactions that canbe prevented by the use of humanized monoclonal antibodies,antibody-based therapies act as pharmacokinetic antagonists which givesthem several important advantages over treatment with more conventionalreceptor antagonists. Firstly, there is no receptor antagonist ford-methamphetamine effects at any of its sites of action in the CNS. Oneof the limitations of development of receptor antagonists is that theywill only be capable of attenuating the effects at one type of receptor.Most drugs of abuse have multiple sites of action. Secondly, unlikeconventional receptor antagonists (or agonists), antibodies do notinhibit the actions of normal endogenous ligands. In fact, it could beargued that removal of the drug by antibodies might allow for a morenormal recovery than treatment with a chemically-derived small moleculecompetitive agonist or antagonist. Thirdly, since antibodies (and theirderivatives like Fab) have extremely high affinities and do not crossthe blood-brain barrier, they actually lower drug concentrationsthroughout the CNS. This allows for a rapid, neuroprotective effect atall sites of action in the CNS.

While monoclonal antibody (mAb) therapy could be a viable approach forantagonizing d-methamphetamine effects, several factors complicate thedesign of antibody therapy for this drug. First, knowledge of therelationship between antibody affinity and therapeutic efficacy islimited. Previous studies have shown that a single dose of ahigh-affinity anti-phencyclidine mAb Fab fragment (K_(D)=1.8 nM) is veryeffective at reversing a phencyclidine-induced overdose in rats(Valentine et al., 1996; Hardin et al., 1998), and that the intact IgG(K_(D)=1.3 nM) can produce long-term reductions in brain phencyclidineconcentrations (Proksch et al., 2000). While these preclinical data forphencyclidine are impressive, the optimal conditions for achieving suchprofound effects are not clear. Second, unlike most drugs of abuse,d-methamphetamine (d-METH) has a major active metabolite, d-amphetamine(d-AMP). This metabolite is present at significant concentrations instudy rats, and d-AMP area under the concentration-versus-time curves(AUC) constitutes 30% and 26% of the total AUC for d-METH and d-AMP inserum and brain, respectively (Riviere et al., 2000). Thus,pharmacological effects due to d-METH and d-AMP may need to be treatedin rats. In humans, however, d-AMP's AUC is <15% of the total serum AUCof d-METH and d-AMP (Cook et al., 1993). Therefore, d-AMP may notcontribute as significantly to d-METH's effects in humans.

Thus, the prior art is deficient in the lack of effective means oftreating d-methamphetamine overdose and addiction by antibody-basedtherapy. The present invention fulfills this long-standing need anddesire in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the recited features, advantages and objectsof the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the use of a long-acting anti-METH antibody medication forthe treatment of drug addiction. In this clinical scenario, the patienthas entered a drug treatment program for d-methamphetamine addiction andthey are treated with a long acting anti-methamphetamine monoclonalantibody-based medication. In this example, the prototype long actingantagonist is an IgG antibody.

FIG. 1A shows the patient's brain after receiving a dose of along-acting anti-METH antibody when they enter into a drug treatmentprogram. This medication will serve as a treatment to help prevent orblunt the rewarding effects of methamphetamine-like drug usage by therecovering addict.

FIG. 1B shows after the patient is pre-treated with theanti-methamphetamine antibody medication, accidental or purposeful useof d-methamphetamine-like drugs by the patient will be blocked or atleast significantly blunted. Thus, the drug(s) cannot reach their siteof action in the central nervous system. This will prevent the patientfrom feeling the reinforcing effects of drug use, and aid in theprevention of relapse.

FIG. 2 shows the use of short-acting anti-methamphetamine monoclonalantibody fragment to treat an overdose resulting fromd-methamphetamine-like drugs. In this case d-methamphetamine is used asthe prototype drug and an anti-methamphetamine monoclonal Fab is used asthe antibody-based medication.

FIG. 2A shows that before the treatment with anti-METH antibodymedication, the patient arrives in an emergency room with a high bodyburden of methamphetamine. This drawing shows that brain concentrationsare very high and the drug is occupying drug sites of action in thepatient's brain. This is producing the clinical overdose.

FIG. 2B shows after treatment with anti-methamphetamine antibodyfragments, the drug is rapidly removed from the brain and the patientquickly recovers. In this example, the prototype short acting antibodybased medication is anti-methamphetamine antibody fragments

FIG. 3 shows the structures of methamphetamine-like stimulants.

FIGS. 4A-4C show the hapten 10 to hapten 24 designed for generatingantibodies that are specific for methamphetamine-like stimulants.

FIG. 5 shows a method of using activated ester to couple the hapten to aprotein to make the antibody. Similar chemistry would apply to all otherstructures shown in FIGS. 4A-4C.

FIG. 6 shows the scheme of synthesis for3-(5′-carboxypentyloxy)methamphetamine hydrochloride.

FIG. 7 shows radioimmunoassay cross-reactivity studies. The two doseresponse curves with squares for symbols show antiserum generated fromimmunization of two different rabbits with a (+)METH PO6 hapten (hapten10 in FIG. 4A with X=5 and connected at the 4 position). The two curveswith circles for symbols show antiserum generated from immunization witha (+)METH MO6 hapten (hapten 10 in FIG. 4A with X=5 and connected at the3 position). The inhibition of [³H]-methamphetamine binding for bothantiserum was tested using both (+)amphetamine and (+)methamphetamine.These data show that careful hapten design can lead to antiserum thathas significant cross-reactivity with both (+)amphetamine and(+)methamphetamine.

FIG. 8 shows the cross-reactivity profile of mAb6H4 determined byradioimmunoassay similar to that described by Owens et al. (1988). Thepercentage bound equals the percentage of B₀ (amount of³H-(+)-methamphetamine binding in the absence of any competing unlabeleddrug) corrected for nonspecific binding. The data were fit with the useof sigmoidal curves that allowed the determination of the IC₅₀ values(concentrations of unlabeled drug that caused a 50% inhibition of³H-(+)-methamphetamine binding).

FIG. 9 compares the ability of a high (left panel) and a low affinity(right panel) mAb to reverse (+)-methamphetamine-induced stimulanteffects (distance traveled and rearing behavior). Before the mAbexperiments, the rats were treated on two separate days with a 1-mg/kgi.v. dose of (+)-methamphetamine followed 30 min later by buffer toestablish control values (data not shown). For the mAb experiments, ratsreceived another 1-mg/kg i.v. (+)-methamphetamine dose followed 30 minlater by a high-affinity mAb or a low-affinity mAb. The data are shownas the mean +1 S.D. (n=6 per group). The arrows and percentages reflectthe degree of mAb-induced reduction in (+)-methamphetamine behavioralresponse compared to control values.

FIG. 10 shows the time course of (+)-methamphetamine-induced distancetraveled in rats (n=6) with either buffer (left panel; open circles) ormAb6H4 (right panel; filled circles) treatment. The i.v.(+)-methamphetamine doses were 0.3, 1.0, and 3.0 mg/kg. The left arrowindicates the time of (+)-methamphetamine administration, and thearrowhead indicates the time of mAb6H4 administration. Shading indicatesthe duration of drug action above saline-induced (baseline) locomotoractivity following buffer or mAb administration. The time needed toreturn to baseline was determined by statistical comparison of thebehavior starting at t=30 min (time of treatment) with each animal'spredosing behavior from −30 min to t=0.

FIG. 11 shows the summary of dose-response results in groups of animalsreceiving 0.3, 1.0, or 3.0 mg/kg (+)-methamphetamine. The animalsreceived saline followed 30 min later by buffer. Three days later, theyreceived a priming dose (0.3, 1.0, or 3.0 mg/kg) of (+)-methamphetaminefollowed 30 min later by buffer (data not shown). This was followed 3days later by a second (+)-methamphetamine priming dose with buffer att=30 min. Three days later, they received a final (+)-methamphetaminedose (0.3, 1.0, or 3.0 mg/kg) followed at t=30 min by mAb6H4 (thehigh-affinity mAb). These data are shown as the means +1 S.D. (n=6 pergroup). * indicates a significant decrease in locomotor activitycompared with (+)-methamphetamine; † indicates a significant increase inlocomotor activity compared with (+)-methamphetamine (p<0.05).

FIG. 12 shows average concentration-versus-time profiles for(+)-methamphetamine and (+)-amphetamine in serum (top panel) and brain(lower panel). The solid lines associated with the (+)-methamphetamineand (+)-amphetamine data show the linear-regression fit to the terminallog concentration-versus-time data as determined by model-independentanalysis. All values are represented as the mean ±1 S.D. (n=3 per timepoint).

FIG. 13 shows average concentration-versus-time profiles for(+)-methamphetamine with (filled circle) and without (open circle)mAb6H4 administered at t=30 min in serum (top panel) and brain (lowerpanel). The solid lines associated with the data show thelinear-regression fit to the terminal log concentration-versus-time dataas determined by model-independent analysis. The arrow indicates thetime of mAb administration. All values are represented as the mean ±1S.D, n=3 per time point. * indicates a significant difference fromcontrol (p<0.05).

FIG. 14 shows average concentration-versus-time profiles for(+)-amphetamine with (filled circles) and without (open circles) mAb6H4administration at t=30 min in serum (top panel) and brain (lower panel).The solid lines associated with the data show the linear-regression fitto the terminal log concentration-versus-time data as determined bymodel-independent analysis. The arrow indicates the time of mAbadministration. All values are represented as the mean ±1 S.D. (n=3 pertime point). * indicates a significant difference from control (p<0.05).

FIG. 15 shows the results from treatment of a methamphetamine-inducedoverdose in rats with a monoclonal anti-methamphetamine antibody oranti-phencyclidine monoclonal antibody (a control monoclonal antibodythat does not bind amphetamine like drugs). Rats (n=4 per group) wereadministered saline (left most bar) or 1.0 mg/kg d-methamphetamine as anintravenous bolus dose. When drug effects were maximizing at 30 minutes,they were treated with saline (control), an anti-phencyclidinemonoclonal antibody Fab fragment (anti-PCP), or ananti-d-methamphetamine-specific monoclonal antibody Fab fragment(anti-methamphetamine).

FIG. 15A shows the distance travel from 30 minutes after saline ord-methamphetamine administration until the end of the experiment, 4.5hours later. *p<0.05 compared to a 1 mg/kg dose of methamphetaminefollowed by the saline control treatment. †p<0.05 compared to a 1 mg/kgdose of methamphetamine followed by the anti-methamphetamine Fabtreatment.

FIG. 15B shows the number of methamphetamine-induced rearing events from30 minutes after saline or d-methamphetamine administration until theend of the experiment, 4.5 hours later. At the 30 min the animals weretreated with ether saline, anti-phencyclidine Fab oranti-d-methamphetamine Fab. *p<0.05 compared to a 1 mg/kg dose ofmethamphetamine followed by the saline control treatment.

FIG. 16 shows the use of anti-(+)METH monoclonal antibodies as apretreatment to reduce drug effects (administered the day before a 1mg/kg methamphetamine challenge dose). The anti-(+) methamphetaminemonoclonal antibody significantly (P<0.05) reduced (+)methamphetamineinduced effects by 42% for distance traveled (top panel) and by 51% forrearing events (bottom panel).

FIG. 17 shows cumulative i.p. dose-response curves for(+)-methamphetamine, (+)-amphetamine and cocaine in rats trained todiscriminate 10 mg/kg cocaine from saline (Table 2, Rat Group III).Abscissa: Mg/kg dose on a log scale. Ordinate: Percentage of responseson the drug (cocaine) key. Each point on the (+)-methamphetamine and(+)-amphetamine dose-response curves represents single observations inthe same four rats, while points on the cocaine dose-response curvesrepresent duplicate observations in the same rats. Brackets at TRAININGshow +/− one standard deviation around mean for six training sessionswith saline and six training sessions with cocaine after respondingstabilized.

FIG. 18 shows cumulative dose-response curves for i.v. and i.p.(+)-methamphetamine in rats. Abscissa: Mg/kg (+)-methamphetamine on alog scale. Ordinate: Percentage of responses on the drug key. Each pointrepresents single observations in each of eleven rats. The rats weretrained to discriminate between saline and 10 mg/kg cocaine, or 5 mg/kgcocaine, or 1 or 3 mg/kg (+)-methamphetamine (see Table 2). Brackets atTRAINING represent six training sessions after saline and six trainingsessions after drug after responding stabilized.

FIG. 19 shows dose-response curves for i.v. (+)-methamphetamine beforeand 1 day after treatment with 1 g/kg of the mAb6H8 antibody in rats(top panel) or before and 4 and 7 days after the antibody treatment(bottom panel). Abscissa: Mg/kg (+)-methamphetamine on a log scale.Ordinate: percentage of responses on the drug key. All 7 rats fromGroups I and II (Table 2) contributed to the dose-response curves in thetop panel, but only 5 of these rats contributed to the data in thebottom panel. Brackets at TRAINING represent six training sessions aftersaline and six training sessions after drug after responding stabilized.

FIG. 20 shows dose-response curves for i.p. (+)-methamphetamine beforeand 4 days (top panel) or 7 days (bottom panel) after treatment with 1g/kg of the mAb6H8 in rats. Abscissa: Mg/kg (+)-methamphetamine on a logscale. Ordinate: Percentage of responses on the drug key. All 7 ratsfrom Groups I and II (Table 2) contributed to the data in the bottompanel, but only 5 of these rats contributed to the data in the toppanel. Brackets at TRAINING represent six training sessions after salineand six training sessions after drug after responding stabilized.

FIG. 21 shows cumulative i.m. dose-response curves for(+)-methamphetamine, (+)-amphetamine and cocaine in pigeons trained todiscriminate 2 or 3 mg/kg (+)-amphetamine from saline. Abscissa: Mg/kgdose on a log scale. Ordinate: Percentage of responses on the drug[(+)-amphetamine] key. Each point on the dose-response curves representsduplicate observations in the pigeons in Group I and single observationsin pigeons in Group II (Table 2), except at the highest dose of eachdrug where only 3 or 4 birds responded. Brackets at TRAINING show +/−onestandard deviation around mean for six training sessions with saline andsix training session with cocaine after responding stabilized.

FIG. 22 shows dose-response curves for i.m. (+)-methamphetamine beforeand 1 and 2, or 7 and 8 days after treatment with 1 g/kg of mAb6H8 inpigeons. Abscissa: Mg/kg (+)-methamphetamine on a log scale. Ordinate:Percentage of responses on the drug key. Two pigeons from Pigeon Group 1and four pigeons from Pigeon Group 2 contributed to these experiments.Brackets at TRAINING represent six training sessions after saline andsix training sessions after drug after responding stabilized.

FIG. 23 shows dose-response curves for intramuscular (+)-amphetaminebefore and 2 or 7 days after treatment with 1 g/kg of mAb6H8 in pigeons.Abscissa: Mg/kg (+)-amphetamine on a log scale. Ordinate: Percentage ofresponses on the drug key. Two pigeons from Pigeon Group 1 and fourpigeons from Pigeon Group 2 contributed to these experiments. Bracketsat TRAINING represent six training sessions after saline and sixtraining sessions after drug after responding stabilized.

FIG. 24 shows dose-response curves for cocaine administered by i.v. andi.p. routes (rats, top panel) or i.m. route (pigeons, bottom panel)before and 1 day after administration of mAb6H8. Abscissa: Mg/kg cocaineon a log scale. Ordinate: Percentage of responses on the drug key. Eachpoint on the dose-response curves represents single observations in twoanimals from the Rat III group or Pigeon II group. Brackets at TRAININGrepresent six training sessions after saline and six training sessionsfor these same subjects.

FIG. 25 shows dose-response curves for i.v. (+)-methamphetamine 1 day(top panel) or 4 and 7 days after the administration of mAb6H4 in rats.Abscissa: Mg/kg (+)-methamphetamine on a log scale. Ordinate: Percentageof responses on the drug key. Brackets at TRAINING show +/− one standarddeviation around mean for six training sessions with saline and sixtraining session with cocaine after responding stabilized.

FIG. 26 shows dose-response curves for intramuscular (+)-methamphetaminebefore and 1 day after 1 g/kg of mAb6H4 in pigeons. Abscissa: Mg/kg(+)-methamphetamine on a log scale. Ordinate: Percentage of responses onthe drug key. Three pigeons from Pigeon Group 2 contributed to theseexperiments. Brackets at TRAINING show +/− one standard deviation aroundmean for six training sessions with saline and six training session with(+)-amphetamine after responding stabilized.

FIG. 27 presents amino acid sequence alignments of the variable regionsof five moderate to high affinity anti-METH and anti-METH/AMP mAb. PanelA presents the amino acid sequences of the heavy chains. Panel B presentthe amino acid sequences of the light chains. The sequences arepresented in single letter amino acid notation and numbered according toKabat and Wu (1991 J Immunol 147:1709-1719). Location of the framework(FR) and CDR residues are indicated for the heavy chains and lightchains.

FIG. 28 presents molecular models of three anti-METH mAb. Upper panel:Stereo view of superimposed molecular models of anti-METH mAb. Thevariable regions of the three mAb were modeled, structurally aligned andrepresented in cartoon format. The framework residues are represented inblue. The CDR regions are colored according to mAb: mAb6H4, blue;mAb6H8, red; mAb4G9, green. The heavy chain, light chain, and CDRregions are labeled. Lower panel: RMSD (Å) of CDRs from the main chainconformation of mAb6H4.

FIG. 29 illustrates modeled structures of the anti-METH mAb variablechains. In this model, METH (magenta) has been computationally dockedinto a pocket at the interface of the VH and VL chains with FlexXsoftware. Left panels: Surface rendering of deep pocket in mAb6H4 andmAb4G9. The VL chain domain is on the left side in blue and the VH chaindomain is on the right in green. Right panels: Stick representation ofmAb6H4 and mAb4G9. Only side chains within 8 angstroms of the METHmolecule are shown for clarity. The view is oriented in a “top view”with the same color scheme as in left panel. The side chains are labeledand numbered in the Kabat scheme as in FIG. 27.

FIG. 30 illustrates immune responses to (+)METH P6 hapten-KLH conjugate.Comparison of rat anti-(+)METH antibody titers by ELISA. KLH immunizedanimals (▪); (+)METH P6-KLH immunized animals (●); and (+)METH P6-KLHimmunized animals with repeated 3 mg/kg, ip (+)METH challenges (◯). (A)day 0 after immunization, (B) day 11 after immunization, (C) day 32after immunization, (D) day 53 after immunization.

FIG. 31 presents rat serum antibody affinities for (+)METH as determinedby ELISA. (+)METH P6-KLH immunized rats (●); (+)METH P6-KLH immunizedrats with repeated (+)METH challenges (◯). (A) day 11 afterimmunization, (B) day 32 after immunization, (C) day 53 afterimmunization.

FIG. 32 illustrates clearance of brain (+)METH. Average concentrationvs. time profiles for (+)METH in the brain without mAb6H4 (•), in anoverdose (□), and in a pretreatment model (∘). The * indicates that boththe overdose and pretreatment points are statistically different fromcontrol (P<0.05). The † indicates that only the pretreatment time pointsare statistically different from the control (P<0.05).

FIG. 33 presents serum concentration of (+)METH over time. (+)METHconcentrations before (□ symbol with “M” inside) and after treatment(open symbols) with five different anti-(+)METH mAbs (n=3 rats/timepoint) and (+)AMP concentrations before (□ symbol with “A” inside) andafter treatment (filled circles) with mAb4G9. (+)AMP concentrations(filled circles) are shown only for mAb4G9 because the other four mAbsdid not produce long-term increases in (+)AMP concentrations. Thebest-fit line was determined using a weighted two-compartmentpharmacokinetic model.

FIG. 34 presents a diagram illustrating a method of preparing a haptenconjugate with an amide-connection. The method comprises coupling ofMO10 directly to CRM₁₉₇ using1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), or,to provide conjugates with different linkers, the conversion of CRM₁₉₇to TFCS-CRM₁₉₇ by N-(ε-trifluoracetylcaproloxy)succinimide ester (TFCS),then coupling to MO6 and MO10 using EDC to give MO6-TFCS-CRM₁₉₇ orMO10-TFCS-CRM₁₉₇.

FIG. 35 presents a diagram illustrating the preparation of haptencompounds with carboxylic acid-ending groups.

FIG. 36 depicts two graphs showing the effect of mAb6H4 (Panel A) andmAb4G9 (Panel B) on METH (closed circles) and AMP (open circles)concentrations in rat serum. METH was infused using a subcutaneousosmotic minipump and once steady state concentration were reached,anti-METH mAb was administered. Rats were sacrificed at various timepoints for determination of serum METH and AMP concentrations. mAb6H4significantly increased METH serum concentrations at the 1, 5, 30, 75,and 120 min, but not at 24 hr, compared to the pre-mAb levels. AMPconcentrations were increased briefly at the 1 and 5 min time points.MAb4G9 significantly increased METH and AMP serum concentrations at alltime points.

FIG. 37 depicts two graphs showing the effect of mAb6H4 (Panel A) andmAb4G9 (Panel B) on METH (closed circles) and AMP (open circles)concentrations in the rat brain. METH was infused using a subcutaneousosmotic minipump and once steady state concentrations were reached,anti-METH mAb was administered. Rats were sacrificed at various timepoints for determination of brain METH and AMP concentrations. METHbrain concentrations were decreased only at the 5 min time point aftermAb6H4 treatment, compared with pre-mAb levels. mAb6H4 had no effect onAMP brain concentrations. mAb4G9 significantly decreased METH and AMPserum concentrations at the 5 and 30 min time points, compared withpre-mAb levels; however, METH and AMP brain concentrations returned topre-mAb levels by 75 min post-treatment.

SUMMARY OF THE INVENTION

One aspect of the invention encompasses a hapten compound of

wherein:

-   -   R₁ is selected from the group consisting of hydrogen and methyl;        and    -   L comprises a linker of at most 10 contiguous atoms, the atoms        being selected from the group consisting of hydrogen,        hydrocarbyl, and substituted hydrocarbyl.

Another aspect of the invention encompasses a hapten compound of formula(II):

wherein:

-   -   R₁ and L are as described for hapten compounds corresponding to        formula (I);    -   n is an integer greater than or equal to 2;    -   X is a carrier molecule that elicits an immunogenic response;        and    -   R₃ is selected from the group consisting of a direct bond,        hydrocarbyl, and substituted hydrocarbyl.

Yet another aspect of the invention encompasses a hapten compound offormula (III):

wherein:

-   -   R₁ and L is as described for hapten compounds corresponding to        formula (I); and    -   R₂ is a heteroatom.

Still another aspect of the invention encompasses a hapten compound offormula (IV):

wherein:

-   -   R₁ and L is as described for hapten compounds corresponding to        formula (I);    -   R₂ is a heteroatom as described in formula (III); and    -   N, X, and R₃ are described in formula (II).

Still yet another aspect of the invention encompasses a composition. Thecomposition may comprise a compound selected from the group consistingof a compound of formula (I), a compound of formula (II), a compound offormula (III), and a compound of formula (IV).

Other aspects of the invention encompass a method for eliciting animmune response. The method comprises administering a composition to thesubject. The composition may comprise a compound selected from the groupconsisting of a compound of formula (I), a compound of formula (II), acompound of formula (III), and a compound of formula (IV).

Still other aspects of the invention encompass a method for generatingspecific antibodies for a compound. The compound may be selected fromthe group consisting of a compound of formula (I), a compound of formula(II), a compound of formula (III), and a compound of formula (IV). Themethod comprises administering the compound to a subject.

Yet other aspects of the invention encompass a method of treating drugaddition. The method comprises eliciting an immune response in adrug-addicted subject by administering a composition to a subject. Thecomposition may comprise a compound selected from the group consistingof a compound of formula (I), a compound of formula (II), a compound offormula (III), and a compound of formula (IV). The immune responseelicited decreases the concentration of (+)methamphetamine,(+)amphetamine, or (+)MDMA in the brain of the subject.

DETAILED DESCRIPTION OF THE INVENTION

Part 1:

This invention encompasses a method of generating high affinitymonoclonal antibodies and their antigen binding fragments (e.g., Fab)for use in treating the medical problems associated with stimulant drugabuse. d-Methamphetamine is the prototypic stimulant molecule because ithas severe addiction liability and produces significant acute andchronic medical problems. Anti-methamphetamine monoclonal antibody (ofany mammalian source) can be used as a prototypic, long acting stimulantantagonist for treating addiction (FIG. 1). In contrast, smallermolecular weight fragments (like Fab) can be used as a prototypicshorter acting, less antigenic, more rapidly eliminated antagonist fortreating drug overdose (FIG. 2). Since intact antibody and smallerfragments like Fab are cleared by different organ systems, this approachwill also provide a greater potential for altering and controlling theendogenous clearance and biological safety of these proteins.

The current studies disclose the development of anti-d-methamphetaminemonoclonal antibodies (mAb) and test the mAb-based therapy in a ratmodel of d-methamphetamine ((+)-METH) overdose. For all studies, thedose of monoclonal antibody was equimolar in binding sites to the bodyburden of a 1-mg/kg intravenous (+)-METH dose at 30 min. The resultsshowed that a (+)-METH-specific, high-affinity murine mAb (K_(D)=11 nM)was 2-3 times more effective than a low-affinity mAb (K_(D)=250 nM) atantagonizing (+)-METH-induced locomotor effects. The high-affinity mAbcompletely reversed locomotor effects produced by 0.3 mg/kg dose of(+)-METH and decreased effects due to 1 mg/kg dose by >60-70%. It wasalso shown that anti-(+)-METH mAbs antagonize (+)-METH effects byaltering brain distribution of (+)-METH. For these studies, ratsreceived 1 mg/kg (+)-METH followed by no treatment (control group) orthe mAb at 30 min. The areas under the (+)-METHconcentration-versus-time curves showed that the mAb increased the serumarea by >9,000% and decreased the brain area by >70%. (+)-Amphetamineserum and brain concentrations were only minimally affected.

Drugs can function as discriminative stimuli to control responding. Thisfunction may overlap with the reinforcing stimuli that are produced bythe drugs, which in turn can contribute to their self administration andabuse liability. Some investigators suggest that the discriminativestimulus properties of drugs may be related to their subjective effectsin humans, although evidence suggests that this relationship iscomplicated. The discriminative stimulus effects of a drug are oftenshared among drugs that produce similar pharmacological effects; andsome investigators suggest that the shared discriminative-stimuluseffects of drugs can be used to define drug classes. For these reasons,drug discrimination has been of great interest to drug abuseresearchers.

If the mAbs disclosed herein bind to d-methamphetamine with highaffinity, they should prevent the drug from penetrating into the centralnervous system to produce its discriminative stimulus effects. If ananti-d-methamphetamine antibody could alter the d-methamphetaminedose-response curve for the discriminative stimulus effects ofd-methamphetamine, it would suggest possible therapeutic usefulness ofantibody treatment for methamphetamine abuse.

Rats and pigeons were trained to discriminate (+)-methamphetamine(rats), cocaine (rats), or (+)-amphetamine (pigeons) from saline afterwhich dose-response curves were determined for (+)-methamphetamine andother drugs before and after administration of a (+)-methamphetaminespecific monoclonal antibody (mAb6H8) (K_(D)=250 nM). Intravenous(+)-methamphetamine was about 3 times more potent as a discriminativestimulus in rats than intraperitoneal (+)-amphetamine. Also in rats,(+)-methamphetamine and (+)-amphetamine were about equipotent asdiscriminative stimuli and were about 3 times more potent than cocaine.In pigeons, (+)-methamphetamine and (+)-amphetamine were nearlyequipotent, while cocaine was slightly less potent. In rats,administration of 1 g/kg of the antibody shifted both intravenous andintraperitoneal dose-response curves for (+)-methamphetaminediscrimination approximately 3-fold to the right for up to 7 days. Asimilar shift of approximately 3-fold to the right that lasted for atleast 7 days occurred when the 1 g/kg dose of the antibody was given topigeons. The antibody did not affect the (+)-amphetamine or cocainedose-response curves. The effects of a second (+)-methamphetaminemonoclonal antibody (mAb6H4)(K_(D)=10 nM) also were studied in both ratsand pigeons. The higher affinity antibody produced a 3-10 fold shift tothe right of the (+)-methamphetamine dose-response curve for drugdiscrimination in both species. These data showed that(+)-methamphetamine-specific antibodies can produce an antagonism of aneffect of (+)-methamphetamine that is closely associated with its abuse.

As used herein, the term “monoclonal antibody” means an antibodycomposition recognizing a discrete antigen determinant. It is notintended to be limited with regard to the source of the antibody or themanner in which it is made. The term antibody is also intended toencompass whole antibodies, biologically functional fragments thereof,chimeric, humanized, and human antibodies comprising portions from morethan one species, or other molecules whose binding properties arederived from antibody-like high affinity binding sites.

In this instance, monoclonal antibodies were produced by hybridomas.However, monoclonal Fab fragments and IgG fragments can also be producedby other methods, for example by using bacteriophage to display andselect polypeptide chains expressed from a V-gene library or geneticengineering.

Biologically functional antibody fragments are those fragmentssufficient for binding to the desired stimulant drug, such as Fab, Fv,F(ab′)₂, sFv, scFv (single-chain antigen-binding protein), and scAbfragments. One can choose among these or whole antibodies for theproperties appropriate to a particular method.

Chimeric antibodies can comprise proteins derived from two differentspecies. The portions derived from two different species can be joinedtogether chemically by conventional techniques or can be prepared as asingle contiguous protein using genetic engineering techniques (See e.g.Cabilly et al., U.S. Pat. No. 4,816,567; Neuberger et al., WO 86/01533and Winter, EP 0,239,400). Such engineered antibodies can be, forinstance, a chimeric antibody comprising murine variable regions andhuman constant regions, or complementarity determining regions(CDR)-grafted antibodies (Tempest et al., 1991). The constant regiondomains can be chosen to have an isotype most suitable for the intendedapplication of the antibodies.

It is contemplated that pharmaceutical compositions may be preparedusing the antibodies of the present invention. In such a case, thepharmaceutical composition comprises the monoclonal antibodies orantigen-binding fragments thereof of the present invention and apharmaceutically acceptable carrier. The present invention has includedthe calculation and administration of equimolar amount of antibodies,and a person having ordinary skill in this art would readily be able todetermine, without undue experimentation, the appropriate dosages androutes of administration of the monoclonal antibodies of the presentinvention. When used in vivo for therapy, the monoclonal antibodies ofthe present invention are administered to the patient or an animal intherapeutically effective amounts, i.e., amounts that eliminate orreduce the effects of stimulant drug overdose or abuse.

In addition to the obvious benefits of a new therapeutic approach, otherimportant contributions would be derived from the present invention. Inas much as the binding properties of receptors and antibodies aresimilar in many ways, the careful design of amphetamine-like haptenscould lead to the selection of antibodies that mimic aspects of theendogenous binding sites of these drugs in the CNS. Molecular studies ofthese antibody binding sites (through protein sequencing,structure-activity studies and molecular modeling) could aid in theprediction of the characteristics necessary for drug-receptorinteraction including neuronal transporters, vesicular storage systems,and with monoamine oxidase. Molecular studies of the sequence of theantibody binding site and the neuronal transporters may also yieldimportant clues concerning the structural rules for molecularinteractions of biologically active compounds. Furthermore, the use ofthese antibody models for screening peptide and organic combinatoriallibraries could lead to discovery of novel agonists or antagonists forthese neuronal transporters.

The present invention is directed to a monoclonal antibody or an antigenbinding fragment thereof that specifically recognizes a stimulant drugof abuse or a metabolite thereof. Representative drugs of abuse or suchmetabolites include d-methamphetamine, d-amphetamine,3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine andstructurally-related analogs of these compounds. In one form, theantibody is of murine origin. Alternatively, the antibody is of humanorigin or contains portions of a human antibody.

As used herein, “structurally-related analogs” refers to any known orunknown chemical moiety (including drug metabolites) that has a similarchemical structure, and similar pharmacological effects (e.g.,behavioral and receptor binding effects) to other knownd-methamphetamine-like drugs. For example, phencyclidine (a differentdrug of abuse) has structurally related analogs likeN-ethyl-1-phenylcyclohexylamine (PCE) and 1-[1-(2-thienyl]piperidine(TCP), which are also drugs of abuse (see Owens et al. 1988 for asimilar discussion for developing anti-PCP antibodies which recognizestructural and pharmacologic similarities in drugs of abuse). Morphinehas structurally related analogs like heroin, which is also abused.Fentanyl has a number of structurally related analogs, which have beenused as drugs of abuse. In the cases phencyclidine and fentanyl thesestructurally related drugs are sometimes referred to as designer drugs,because they mimic the effects which are desired by drug abusers.

The present invention also provides methods of treating stimulant drugabuse or overdose, comprising the step of administering apharmacological effective dose of the monoclonal antibody or an antigenbinding fragment thereof of the present invention to an individual inneed of such treatment. Representative stimulant drugs are describedabove.

The present invention is also directed to a method of generating aclass-specific monoclonal antibody that recognizes methamphetamine-likestimulants, comprising the step of: immunizing animals with thesubstituted methamphetamines or hydrochlorides thereof disclosed herein.

Part 2:

The present invention is also directed to hapten compounds.

I. Hapten Compounds

One aspect of the invention encompasses a hapten compound that compriseseither (+) methamphetamine or (+) amphetamine conjugated to a linker.Generally speaking, the hapten compound is designed to elicit an immuneresponse in a subject that generates antibodies that recognize one ormore of (+) methamphetamine, (+) amphetamine, or (+)3,4-methylenedioxymethamphetamine ((+) MDMA). In certain embodiments,the hapten compound is designed to generate antibodies that recognize atleast two compounds from the group consisting of (+) methamphetamine,(+) amphetamine, and (+) MDMA. In an exemplary embodiment, the compoundis designed to generate antibodies that recognize all three compounds ofthe group consisting of (+) methamphetamine, (+) amphetamine, and (+)MDMA.

In one embodiment, the hapten compound has formula (I):

wherein:

-   -   R₁ is hydrogen or a methyl; and    -   L is a linker.

In some embodiments, R₁ is hydrogen (i.e., forming (+) amphetamine). Inother embodiments, R₁ is a methyl group (i.e., forming (+)methamphetamine).

In general, L is comprised of atoms so as to facilitate an orientationof the (+) methamphetamine or (+) amphetamine sufficient to generatedesired antibodies. In this context, “desired” antibodies includeantibodies that recognize (+) methamphetamine, (+) amphetamine, or (+)MDMA. L is also typically not strongly immunogenic. In other words, Lmay be designed so that antibodies generated against a compound of theinvention recognize the compound and not merely L.

Furthermore, in exemplary embodiments, L is designed to generateantibodies with a long functional half-life. For more details, see theexamples.

The length of L may be expressed as the number of contiguous atomsforming the shortest path from one substructure that L connects to theother substructure. Typically, L may not be longer than 10 contiguousatoms. In one embodiment, L is at least 2, but not more than 10contiguous atoms in length. In another embodiment, L may be 2, 3, 4, 5,6, 7, 8, 9, or 10 contiguous atoms in length. In yet another embodiment,L may be about 5 to 10 contiguous atoms.

As will be appreciated by a skilled artisan, the atoms comprising L mayvary widely. Typically, the atoms impart the appropriate degree offlexibility, as detailed above. Suitable atoms forming L may be selectedfrom the group comprising hydrogen, hydrocarbyl, substitutedhydrocarbyls, and heteroatoms. In some embodiments, L may be comprisedof amino acids, such as glycine or proline. In other embodiments, L maybe comprised of nucleotides. In further embodiments, L may be linear,branched, or may comprise ring structures.

It is also envisioned that L may be attached to the benzene ring of (+)methamphetamine or (+) amphetamine at a variety of positions withoutdeparting from the scope of the invention. For example, in oneembodiment, L may be attached at the meta position of the benzene ringas shown in formula (V):

wherein:

-   -   L and R₁ have the same substituents as detailed for compounds        corresponding to formula (I).

In another embodiment, L may be attached at the ortho position as shownin formula (VI):

wherein:

-   -   L and R₁ have the same substituents as detailed for compounds        corresponding to formula (I).

In yet another embodiment, L may be attached at the para position asshown in formula (VII):

wherein:

-   -   L and R₁ have the same substituents as detailed for compounds        corresponding to formula (I).

Exemplary embodiments of L may be comprised of

(CH₂)_(m), wherein m is an integer between about 5 and about 10, whereinm is an integer between about 6 and about 10, wherein m is an integerbetween about 7 and 10; wherein m is an integer between about 8 and 10,wherein m is 9, and wherein m is 10. In other exemplary embodiments Lmay be comprised of

O(CH₂)_(m), wherein m is an integer between about 5 and 9, wherein m isan integer between about 6 and 9, wherein m is an integer between about7 and 9; wherein m is 8, and wherein m is 9. In an alternativeembodiment, L may be comprised of a group listed in Table A.

TABLE A L Group Position on Benzene Ring —(CH₂) Para —(CH₂) Meta —(CH₂)Ortho —(CH₂)₂ Para —(CH₂)₂ Meta —(CH₂)₂ Ortho —(CH₂)₃ Para —(CH₂)₃ Meta—(CH₂)₃ Ortho —(CH₂)₄ Para —(CH₂)₄ Meta —(CH₂)₄ Ortho —(CH₂)₅ Para—(CH₂)₅ Meta —(CH₂)₅ Ortho —(CH₂)₆ Para —(CH₂)₆ Meta —(CH₂)₆ Ortho—(CH₂)₇ Para —(CH₂)₇ Meta —(CH₂)₇ Ortho —(CH₂)₈ Para —(CH₂)₈ Meta—(CH₂)₈ Ortho —(CH₂)₉ Para —(CH₂)₉ Meta —(CH₂)₉ Ortho —(CH₂)₁₀ Para—(CH₂)₁₀ Meta —(CH₂)₁₀ Ortho —O(CH₂) Para —O(CH₂) Meta —O(CH₂) Ortho—O(CH₂)₂ Para —O(CH₂)₂ Meta —O(CH₂)₂ Ortho —O(CH₂)₃ Para —O(CH₂)₃ Meta—O(CH₂)₃ Ortho —O(CH₂)₄ Para —O(CH₂)₄ Meta —O(CH₂)₄ Ortho —O(CH₂)₅ Para—O(CH₂)₅ Meta —O(CH₂)₅ Ortho —O(CH₂)₆ Para —O(CH₂)₆ Meta —O(CH₂)₆ Ortho—O(CH₂)₇ Para —O(CH₂)₇ Meta —O(CH₂)₇ Ortho —O(CH₂)₈ Para —O(CH₂)₈ Meta—O(CH₂)₈ Ortho —O(CH₂)₉ Para —O(CH₂)₉ Meta —O(CH₂)₉ Ortho ^(‡)—X Para^(‡)—X Meta ^(‡)—X Ortho ^(‡)—X₂ Para ^(‡)—X₂ Meta ^(‡)—X₂ Ortho ^(‡)—X₃Para ^(‡)—X₃ Meta ^(‡)—X₃ Ortho ^(‡)—X₄ Para ^(‡)—X₄ Meta ^(‡)—X₄ Ortho^(‡)—X₅ Para ^(‡)—X₅ Meta ^(‡)—X₅ Ortho ^(‡)—X₆ Para ^(‡)—X₆ Meta^(‡)—X₆ Ortho ^(‡)—X₇ Para ^(‡)—X₇ Meta ^(‡)—X₇ Ortho ^(‡)—X₈ Para^(‡)—X₈ Meta ^(‡)—X₈ Ortho ^(‡)—X₉ Para ^(‡)—X₉ Meta ^(‡)—X₉ Ortho^(‡)—X₁₀ Para ^(‡)—X₁₀ Meta ^(‡)—X₁₀ Ortho ^(‡)wherein X may be any atomselected from the group comprising C, O, N, P and S; including theappropriate number of hydrogens to balance charge.

Methods of making hapten compounds of formula (I) are known in the artor are otherwise described herein. For instance, see FIG. 35 depicting ascheme illustrating the preparation of hapten compounds of the inventionwith carboxylic acid-ending groups.

In another embodiment, the compound may have formula (III):

wherein:

-   -   R₁ and L are as described for hapten compounds corresponding to        formula (I); and    -   R₂ may be a heteroatom.

In certain embodiments for compounds corresponding to formula (III), R₂may be a carbon atom, an oxygen atom, a nitrogen atom, a sulfur atom, ora phosphorous atom. In one alternative embodiment, R₂ may be a carbonatom. In another alternative embodiment, R₂ may be an oxygen atom. Inyet another alternative embodiment, R₂ may be a nitrogen atom. In stillyet another alternative embodiment, R₂ may be a phosphorous atom. In anadditional alternative embodiment, R₂ may be a sulfur atom.

In an exemplary embodiment, a compound of formula (III) may have oxygenfor R₂ and (CH₂)₉ as L.

Other exemplary hapten compounds having formula (I) or (III) are shownin Table A.

II. Hapten Compounds Conjugated to Carrier Molecules

In another aspect of the invention, any of the hapten compounds havingformulas (I) or (III) may be conjugated, via a linker, L, to a carriermolecule X. Generally speaking, the carrier molecule is selected so thatit enhances the immunogenicity of the hapten compound. For instance, thecarrier molecule may provide a T-cell epitope to enhance theimmunogenicity of the hapten compound. These compounds may be utilizedfor a variety of suitable uses, including, as a therapeutic immunogeniccompound (described in more detail herein), and to elicit the generationof antibodies that may be utilized in passive therapies or in methods ofpurification or detection.

In one embodiment, a hapten compound corresponding to formula (I) isconjugated via L to a carrier molecule, X, to form a compound havingformula (II):

wherein:

-   -   R₁ and L are as described for hapten compounds corresponding to        formula (I);    -   R₃ may be selected from the group comprising a direct bond,        hydrocarbyl, and substituted hydrocarbyl;    -   X is a carrier molecule that is capable of eliciting an immune        response; and    -   n is an integer greater than or equal to 2.

In one alternative embodiment, R₃ may be a direct bond. In anotheralternative embodiment, R₃ may be a hydrocarbyl. In yet anotheralternative embodiment, R₃ may be a substituted hydrocarbyl. In anexemplary alternative of this embodiment, R₃ may be (CH₂)₄ or(CH₂)₄NHCO(CH₂)₅.

Typically, X may be a protein, lipid, carbohydrate, or any combinationthereof that is capable of eliciting an immune response. For instance,in one embodiment, X may be a polysaccharide, such as mannan. In anotherembodiment, X may be a lipopolysaccharide, such as a lipopolysaccharidederived from Salmonella typhosa.

In exemplary embodiments, X is a protein. In a particular embodiment Xmay be selected from the group of proteins comprising keyhole limpethemocyanin (KLH), ovalbumin, bovine serum albumin (BSA), sheep albumin,thyroglobulin, and any modifications, derivatives, or analogues thereof.For instance, in one embodiment, X may be BSA or cationized BSA. Inanother embodiment, X may be KLH. In yet another embodiment, X may bethyroglobulin. In still yet another embodiment, X may be ovalbumin.

In another particular embodiment, X may be a bacterial toxin or toxoid.Non-limiting examples of suitable bacterial toxins or toxoids mayinclude tetanus toxoid, diphtheria toxoid, non-toxic mutant diphtheriatoxoid CRM₁₉₇, outer membrane protein complex (OMPC) from Neisseriameningitidis, the B subunit of heat-labile Escherichia coli, recombinantexoprotein A from Pseudomonas aeruginosa (rEPA), cholera toxin B-(CTB),pertussis toxin and filamentous hemagglutinin, shiga toxin, and the LTBfamily of bacterial toxins.

In yet another embodiment, X may be a lectin. Non-limiting examples ofsuitable lectins may include ricin-B subunit, abrin and sweet pealectin.

In an alternative embodiment, X may be selected from the groupcomprising retrovirus nucleoprotein (retro NP), rabies ribonucleoprotein(rabies RNP), plant viruses (e.g. TMV, cow pea and cauliflower mosaicviruses), vesicular stomatitis virus-nucleocapsid protein (VSV-N),poxvirus subunits and Semliki forest virus subunits.

In another alternative embodiment, X may be an artificial molecularcarrier. Non-limiting examples of an artificial molecular carrierinclude multiantigenic peptides (MAP) and microspheres. In an additionalembodiment X may be yeast virus-like particles (VLPs). In anotheradditional embodiment, X may be a malarial protein antigen.

Furthermore, X may be selected from the group comprising Diphtheria,Tetanus, and Pertussis vaccines or components thereof; poliovirusvaccines and components thereof; Rubella, Mumps, and Measles vaccines orcomponents thereof; Hepatitis vaccines (A,B,C, and delta) and componentsthereof; Haemophilus (A and B) vaccines and components thereof; vacciniaand smallpox vaccines and components thereof; and varicella-zostervaccines and components thereof.

In a preferred embodiment, X may be a pharmaceutically acceptablecarrier for human subjects. In other words, X may be a carrier thatsafely elicits an antibody response in a subject. In this context, theterm “safely” means that the carrier does not substantially elicit animmune response that cross-reacts with a self-protein, or a regularlyingested protein of the subject. Non-limiting examples ofpharmaceutically acceptable carriers for use in human subjects includemutant diphtheria toxoid (CRM₁₉₇) and tetanus toxoid. In one preferredembodiment, X may be diphtheria toxoid CRM₁₉₇. In another preferredembodiment, X may be tetanus toxoid.

To increase the elicited immune response to a hapten compound of theinvention, generally more than one hapten compound is conjugated to anindividual carrier molecule, X, as expressed by n (i.e., the number ofhapten compounds conjugated to X). Generally speaking, n is an integergreater than or equal to 2. In one embodiment, n may be 2, 3, 4, or 5.In another embodiment, n may be 6, 7, 8, or 9. In an exemplaryembodiment, n is greater than or equal to 5.

In another embodiment, a hapten compound corresponding to formula (III)is conjugated via L to a carrier molecule, X, to form a compound havingformula (IV):

wherein:

-   -   R₁, L, n, and X are as described for compounds corresponding to        formula (II);    -   R₂ is as described for compounds corresponding to formula (III);        and    -   R₃ may be selected from the group comprising a direct bond,        hydrocarbyl, and substituted hydrocarbyl. In an exemplary        alternative of this embodiment, R₃ is (CH₂)₄ or        (CH₂)₄NHCO(CH₂)₅.

Exemplary compounds of the invention having formulas (II) and (IV) areshown in Table B.

TABLE B Position on Benzene L Group Ring Carrier —(CH₂) Para KLH —(CH₂)Para Ovalbumin —(CH₂) Para BSA —(CH₂) Para Diptheria CRM₁₉₇ —(CH₂) ParaTetanus toxoid —(CH₂) Meta KLH —(CH₂ Meta Ovalbumin —(CH₂) Meta BSA—(CH₂) Meta Diptheria CRM₁₉₇ —(CH₂) Meta Tetanus toxoid —(CH₂)₂ Para KLH—(CH₂)₂ Para Ovalbumin —(CH₂)₂ Para BSA —(CH₂)₂ Para Diptheria CRM₁₉₇—(CH₂)₂ Para Tetanus toxoid —(CH₂)₂ Meta KLH —(CH₂)₂ Meta Ovalbumin—(CH₂)₂ Meta BSA —(CH₂)₂ Meta Diptheria CRM₁₉₇ —(CH₂)₂ Meta Tetanustoxoid —(CH₂)₃ Para KLH —(CH₂)₃ Para Ovalbumin —(CH₂)₃ Para BSA —(CH₂)₃Para Diptheria CRM₁₉₇ —(CH₂)₃ Para Tetanus toxoid —(CH₂)₃ Meta KLH—(CH₂)₃ Meta Ovalbumin —(CH₂)₃ Meta BSA —(CH₂)₃ Meta Diptheria CRM₁₉₇—(CH₂)₃ Meta Tetanus toxoid —(CH₂)₄ Para KLH —(CH₂)₄ Para Ovalbumin—(CH₂)₄ Para BSA —(CH₂)₄ Para Diptheria CRM₁₉₇ —(CH₂)₄ Para Tetanustoxoid —(CH₂)₄ Meta KLH —(CH₂)₄ Meta Ovalbumin —(CH₂)₄ Meta BSA —(CH₂)₄Meta Diptheria CRM₁₉₇ —(CH₂)₄ Meta Tetanus toxoid —(CH₂)₅ Para KLH—(CH₂)₅ Para Ovalbumin —(CH₂)₅ Para BSA —(CH₂)₅ Para Diptheria CRM₁₉₇—(CH₂)₅ Para Tetanus toxoid —(CH₂)₅ Meta KLH —(CH₂)₅ Meta Ovalbumin—(CH₂)₅ Meta BSA —(CH₂)₅ Meta Diptheria CRM₁₉₇ —(CH₂)₅ Meta Tetanustoxoid —(CH₂)₆ Para KLH —(CH₂)₆ Para Ovalbumin —(CH₂)₆ Para BSA —(CH₂)₆Para Diptheria CRM₁₉₇ —(CH₂)₆ Para Tetanus toxoid —(CH₂)₆ Meta KLH—(CH₂)₆ Meta Ovalbumin —(CH₂)₆ Meta BSA —(CH₂)₆ Meta Diptheria CRM₁₉₇—(CH₂)₆ Meta Tetanus toxoid —(CH₂)₇ Para KLH —(CH₂)₇ Para Ovalbumin—(CH₂)₇ Para BSA —(CH₂)₇ Para Diptheria CRM₁₉₇ —(CH₂)₇ Para Tetanustoxoid —(CH₂)₇ Meta KLH —(CH₂)₇ Meta Ovalbumin —(CH₂)₇ Meta BSA —(CH₂)₇Meta Diptheria CRM₁₉₇ —(CH₂)₇ Meta Tetanus toxoid —(CH₂)₈ Para KLH—(CH₂)₈ Para Ovalbumin —(CH₂)₈ Para BSA —(CH₂)₈ Para Diptheria CRM₁₉₇—(CH₂)₈ Para Tetanus toxoid —(CH₂)₈ Meta KLH —(CH₂)₈ Meta Ovalbumin—(CH₂)₈ Meta BSA —(CH₂)₈ Meta Diptheria CRM₁₉₇ —(CH₂)₈ Meta Tetanustoxoid —(CH₂)₉ Para KLH —(CH₂)₉ Para Ovalbumin —(CH₂)₉ Para BSA —(CH₂)₉Para Diptheria CRM₁₉₇ —(CH₂)₉ Para Tetanus toxoid —(CH₂)₉ Meta KLH—(CH₂)₉ Meta Ovalbumin —(CH₂)₉ Meta BSA —(CH₂)₉ Meta Diptheria CRM₁₉₇—(CH₂)₉ Meta Tetanus toxoid —(CH₂)₁₀ Para KLH —(CH₂)₁₀ Para Ovalbumin—(CH₂)₁₀ Para BSA —(CH₂)₁₀ Para Diptheria CRM₁₉₇ —(CH₂)₁₀ Para Tetanustoxoid —(CH₂)₁₀ Meta KLH —(CH₂)₁₀ Meta Ovalbumin —(CH₂)₁₀ Meta BSA—(CH₂)₁₀ Meta Diptheria CRM₁₉₇ —(CH₂)₁₀ Meta Tetanus toxoid —O(CH₂) ParaKLH —O(CH₂) Para Ovalbumin —O(CH₂) Para BSA —O(CH₂) Para DiptheriaCRM₁₉₇ —O(CH₂) Para Tetanus toxoid —O(CH₂) Meta KLH —O(CH₂) MetaOvalbumin —O(CH₂) Meta BSA —O(CH₂) Meta Diptheria CRM₁₉₇ —O(CH₂) MetaTetanus toxoid —O(CH₂)₂ Para KLH —O(CH₂)₂ Para Ovalbumin —O(CH₂)₂ ParaBSA —O(CH₂)₂ Para Diptheria CRM₁₉₇ —O(CH₂)₂ Para Tetanus toxoid —O(CH₂)₂Meta KLH —O(CH₂)₂ Meta Ovalbumin —O(CH₂)₂ Meta BSA —O(CH₂)₂ MetaDiptheria CRM₁₉₇ —O(CH₂)₂ Meta Tetanus toxoid —O(CH₂)₃ Para KLH —O(CH₂)₃Para Ovalbumin —O(CH₂)₃ Para BSA —O(CH₂)₃ Para Diptheria CRM₁₉₇ —O(CH₂)₃Para Tetanus toxoid —O(CH₂)₃ Meta KLH —O(CH₂)₃ Meta Ovalbumin —O(CH₂)₃Meta BSA —O(CH₂)₃ Meta Diptheria CRM₁₉₇ —O(CH₂)₃ Meta Tetanus toxoid—O(CH₂)₄ Para KLH —O(CH₂)₄ Para Ovalbumin —O(CH₂)₄ Para BSA —O(CH₂)₄Para Diptheria CRM₁₉₇ —O(CH₂)₄ Para Tetanus toxoid —O(CH₂)₄ Meta KLH—O(CH₂)₄ Meta Ovalbumin —O(CH₂)₄ Meta BSA —O(CH₂)₄ Meta Diptheria CRM₁₉₇—O(CH₂)₄ Meta Tetanus toxoid —O(CH₂)₅ Para KLH —O(CH₂)₅ Para Ovalbumin—O(CH₂)₅ Para BSA —O(CH₂)₅ Para Diptheria CRM₁₉₇ —O(CH₂)₅ Para Tetanustoxoid —O(CH₂)₅ Meta KLH —O(CH₂)₅ Meta Ovalbumin —O(CH₂)₅ Meta BSA—O(CH₂)₅ Meta Diptheria CRM₁₉₇ —O(CH₂)₅ Meta Tetanus toxoid —O(CH₂)₆Para KLH —O(CH₂)₆ Para Ovalbumin —O(CH₂)₆ Para BSA —O(CH₂)₆ ParaDiptheria CRM₁₉₇ —O(CH₂)₆ Para Tetanus toxoid —O(CH₂)₆ Meta KLH —O(CH₂)₆Meta Ovalbumin —O(CH₂)₆ Meta BSA —O(CH₂)₆ Meta Diptheria CRM₁₉₇ —O(CH₂)₆Meta Tetanus toxoid —O(CH₂)₇ Para KLH —O(CH₂)₇ Para Ovalbumin —O(CH₂)₇Para BSA —O(CH₂)₇ Para Diptheria CRM₁₉₇ —O(CH₂)₇ Para Tetanus toxoid—O(CH₂)₇ Meta KLH —O(CH₂)₇ Meta Ovalbumin —O(CH₂)₇ Meta BSA —O(CH₂)₇Meta Diptheria CRM₁₉₇ —O(CH₂)₇ Meta Tetanus toxoid —O(CH₂)₈ Para KLH—O(CH₂)₈ Para Ovalbumin —O(CH₂)₈ Para BSA —O(CH₂)₈ Para Diptheria CRM₁₉₇—O(CH₂)₈ Para Tetanus toxoid —O(CH₂)₈ Meta KLH —O(CH₂)₈ Meta Ovalbumin—O(CH₂)₈ Meta BSA —O(CH₂)₈ Meta Diptheria CRM₁₉₇ —O(CH₂)₈ Meta Tetanustoxoid —O(CH₂)₉ Para KLH —O(CH₂)₉ Para Ovalbumin —O(CH₂)₉ Para BSA—O(CH₂)₉ Para Diptheria CRM₁₉₇ —O(CH₂)₉ Para Tetanus toxoid —O(CH₂)₉Meta KLH —O(CH₂)₉ Meta Ovalbumin —O(CH₂)₉ Meta BSA —O(CH₂)₉ MetaDiptheria CRM₁₉₇ —O(CH₂)₉ Meta Tetanus toxoid ^(‡)—X₁ Para KLH ^(‡)—X₁Para Ovalbumin ^(‡)—X₁ Para BSA ^(‡)—X₁ Para Diptheria CRM₁₉₇ ^(‡)—X₁Para Tetanus toxoid ^(‡)—X₁ Meta KLH ^(‡)—X₁ Meta Ovalbumin ^(‡)—X₁ MetaBSA ^(‡)—X₁ Meta Diptheria CRM₁₉₇ ^(‡)—X₁ Meta Tetanus toxoid ^(‡)—X₂Para KLH ^(‡)—X₂ Para Ovalbumin ^(‡)—X₂ Para BSA ^(‡)—X₂ Para DiptheriaCRM₁₉₇ ^(‡)—X₂ Para Tetanus toxoid ^(‡)—X₂ Meta KLH ^(‡)—X₂ MetaOvalbumin ^(‡)—X₂ Meta BSA ^(‡)—X₂ Meta Diptheria CRM₁₉₇ ^(‡)—X₂ MetaTetanus toxoid ^(‡)—X₃ Para KLH ^(‡)—X₃ Para Ovalbumin ^(‡)—X₃ Para BSA^(‡)—X₃ Para Diptheria CRM₁₉₇ ^(‡)—X₃ Para Tetanus toxoid ^(‡)—X₃ MetaKLH ^(‡)—X₃ Meta Ovalbumin ^(‡)—X₃ Meta BSA ^(‡)—X₃ Meta DiptheriaCRM₁₉₇ ^(‡)—X₃ Meta Tetanus toxoid ^(‡)—X₄ Para KLH ^(‡)—X₄ ParaOvalbumin ^(‡)—X₄ Para BSA ^(‡)—X₄ Para Diptheria CRM₁₉₇ ^(‡)—X₄ ParaTetanus toxoid ^(‡)—X₄ Meta KLH ^(‡)—X₄ Meta Ovalbumin ^(‡)—X₄ Meta BSA^(‡)—X₄ Meta Diptheria CRM₁₉₇ ^(‡)—X₄ Meta Tetanus toxoid ^(‡)—X₅ ParaKLH ^(‡)—X₅ Para Ovalbumin ^(‡)—X₅ Para BSA ^(‡)—X₅ Para DiptheriaCRM₁₉₇ ^(‡)—X₅ Para Tetanus toxoid ^(‡)—X₅ Meta KLH ^(‡)—X₅ MetaOvalbumin ^(‡)—X₅ Meta BSA ^(‡)—X₅ Meta Diptheria CRM₁₉₇ ^(‡)—X₅ MetaTetanus toxoid ^(‡)—X₆ Para KLH ^(‡)—X₆ Para Ovalbumin ^(‡)—X₆ Para BSA^(‡)—X₆ Para Diptheria CRM₁₉₇ ^(‡)—X₆ Para Tetanus toxoid ^(‡)—X₆ MetaKLH ^(‡)—X₆ Meta Ovalbumin ^(‡)—X₆ Meta BSA ^(‡)—X₆ Meta DiptheriaCRM₁₉₇ ^(‡)—X₆ Meta Tetanus toxoid ^(‡)—X₇ Para KLH ^(‡)—X₇ ParaOvalbumin ^(‡)—X₇ Para BSA ^(‡)—X₇ Para Diptheria CRM₁₉₇ ^(‡)—X₇ ParaTetanus toxoid ^(‡)—X₇ Meta KLH ^(‡)—X₇ Meta Ovalbumin ^(‡)—X₇ Meta BSA^(‡)—X₇ Meta Diptheria CRM₁₉₇ ^(‡)—X₇ Meta Tetanus toxoid ^(‡)—X₈ ParaKLH ^(‡)—X₈ Para Ovalbumin ^(‡)—X₈ Para BSA ^(‡)—X₈ Para DiptheriaCRM₁₉₇ ^(‡)—X₈ Para Tetanus toxoid ^(‡)—X₈ Meta KLH ^(‡)—X₈ MetaOvalbumin ^(‡)—X₈ Meta BSA ^(‡)—X₈ Meta Diptheria CRM₁₉₇ ^(‡)—X₈ MetaTetanus toxoid ^(‡)—X₉ Para KLH ^(‡)—X₉ Para Ovalbumin ^(‡)—X₉ Para BSA^(‡)—X₉ Para Diptheria CRM₁₉₇ ^(‡)—X₉ Para Tetanus toxoid ^(‡)—X₉ MetaKLH ^(‡)—X₉ Meta Ovalbumin ^(‡)—X₉ Meta BSA ^(‡)—X₉ Meta DiptheriaCRM₁₉₇ ^(‡)—X₉ Meta Tetanus toxoid ^(‡)—X₁₀ Para KLH ^(‡)—X₁₀ ParaOvalbumin ^(‡)—X₁₀ Para BSA ^(‡)—X₁₀ Para Diptheria CRM₁₉₇ ^(‡)—X₁₀ ParaTetanus toxoid ^(‡)—X₁₀ Meta KLH ^(‡)—X₁₀ Meta Ovalbumin ^(‡)—X₁₀ MetaBSA ^(‡)—X₁₀ Meta Diptheria CRM₁₉₇ ^(‡)—X₁₀ Meta Tetanus toxoid^(‡)wherein X may be any atom selected from the group comprising C, O,N, P and S; including the appropriate number of hydrogens to balancecharge.III. Conjugation Chemistry

The compounds detailed in part (II) corresponding to formulas (I), (II),(III), and (IV) may be made by a variety of methods generally known inthe art or as described herein. Irrespective of the process utilized forconjugation of a hapten compound to a carrier molecule (X) via a linker(L), the process selected will typically result in a compound having arelatively high epitope density, i.e., the number of hapten compoundsconjugated to a single carrier molecule, (X), as expressed above as n.The conjugation method used will depend upon the chemistry of coupling aparticular hapten compound to a molecular carrier. In general, areactive site on a first compound is linked to a reactive site on asecond compound, using a coupling agent or catalyst. An exemplaryprocess is described below in more detail.

Referring to FIG. 34, in an exemplary embodiment to form compoundshaving formula (II) or (IV) the hapten compound may be conjugated viathe linker (L) to a carrier molecule (X) by formation of an amide bond.Generally speaking, the carboxyl group of the linker is reacted with anamide group on the carrier molecule. However, in certain embodiments,the carboxyl group of the linker may be reacted with a second linkerthat is reacted with an amide group on the carrier molecule. Such secondlinkers may be homobifunctional or heterobifunctional linkers, and arewell known in the art. Suitable coupling agents may include EDC,Carbodiimide-HCL, glutaraldehyde and other similar agents. In oneembodiment, L of formula (II) or (III) may be conjugated to X byreaction with EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride) (see FIG. 34). Alternatively, X may be reacted with TFCS,and then reacted with EDC to form an amide bond with L of formula (II)or (IV)(see FIG. 34).

Typically, in a conjugation reaction that is based on an amide bond, theratio of carrier molecule to hapten compound may be between about 1:25and about 1:100. In one embodiment, the ratio may be between about 1:25and about 1:50. In another embodiment, the ratio may be between about1:50 and about 1:75. In yet another embodiment, the ratio may be betweenabout 1:75 and about 1:100. In still another embodiment, the ratio maybe between about 1:30 and about 1:90.

The ratio of carrier molecule to hapten compound should be selected tomaximize the number of hapten compounds conjugated to the molecularcarrier (i.e. n, as described in formulas (II) and (IV). Generally, n isan integer greater than or equal to 2. In one embodiment, n may be 3, 4,or 5. In another embodiment, n may be 6, 7, 8, or 9. In an exemplaryembodiment, n is greater than or equal to 5. The ratio of carriermolecule to hapten compound may be determined by MALDI MS.

IV. Immunogenic Compositions Comprising Hapten Compounds

An additional aspect of the invention encompasses an immunogeniccomposition comprising a hapten compound. In some embodiments, theimmunogenic composition may comprise a hapten compound selected from thegroup consisting of a compound of formula (I), a compound of formula(II), a compound of formula (III), and a compound of formula (IV). Inone embodiment, the immunogenic composition may comprise a compound offormula (I). In another embodiment, the immunogenic composition maycomprise a compound of formula (II). In yet another embodiment, theimmunogenic composition may comprise a compound of formula (III). Instill another embodiment, the immunogenic composition may comprise acompound of formula (IV).

In certain embodiments, an immunogenic composition comprising a haptencompound of the invention may further comprise an adjuvant. Generallyspeaking, an adjuvant may be used to increase the immune response to ahapten compound of the invention. For instance, an adjuvant may be usedto increase antibody affinity, antibody titer, and the duration of theimmune response in a subject. Non-limiting examples of adjuvants includealum, TiterMax Gold, Ribi, ASO4, Freund's complete adjuvant, andFreund's incomplete adjuvant. In one embodiment, the adjuvant may bealum. In another embodiment, the adjuvant may be TiterMax Gold. In yetanother embodiment, the adjuvant may be Ribi. In still anotherembodiment, the adjuvant may be ASO4. In still yet another embodiment,the adjuvant may be Freund's complete adjuvant. In an additionalembodiment, the adjuvant may be Freund's incomplete adjuvant. In anexemplary embodiment, the adjuvant is pharmaceutically acceptable foruse in a human subject. Generally speaking, a pharmaceuticallyacceptable adjuvant is pyrogen free and will not induce anaphylacticshock in a subject. Non-limiting examples of pharmaceutically acceptableadjuvants for use in humans include alum and ASO4.

In some embodiments, an immunogenic composition comprising a haptencompound may further comprise a pharmaceutically acceptable carrier, asdescribed in section II above. Briefly, a pharmaceutically acceptablecarrier safely elicits an antibody response in a subject. In thiscontext, safely means that the carrier does not substantially elicit animmune response that cross-reacts with a self-protein, or a regularlyingested protein of the subject. Those skilled in the art will recognizethat the selection of a pharmaceutically acceptable carrier depends onlarge part on the subject that is administered the carrier.

In certain embodiments, it is envisioned that a particular molecularcarrier may be conjugated to more than one type of hapten compound. Forinstance, a particular molecular carrier may be conjugated to a haptencompound of formula (II) and formula (IV). Alternatively, a particularmolecular carrier may be conjugated to at least two hapten compoundslisted in Table A.

In further embodiments, an immunogenic composition comprising a haptencompound may further comprise an adjuvant and a pharmaceuticallyacceptable carrier. In some embodiments, an immunogenic composition ofthe invention may comprise a combination of an adjuvant and carrierlisted in Table C.

TABLE C Adjuvant Carrier Alum Ovalbumin Alum BSA Alum KLH Alum DiptheriaCRM₁₉₇ Alum Tetanus toxoid Titermax gold Ovalbumin Titermax gold BSATitermax gold KLH Titermax gold Diptheria CRM₁₉₇ Titermax gold Tetanustoxoid ASO4 Ovalbumin ASO4 BSA ASO4 KLH ASO4 Diptheria CRM₁₉₇ ASO4Tetanus toxoid Ribi Ovalbumin Ribi BSA Ribi KLH Ribi Diptheria CRM₁₉₇Ribi Tetanus toxoid Freund's complete adjuvant Ovalbumin Freund'scomplete adjuvant BSA Freund's complete adjuvant KLH Freund's completeadjuvant Diptheria CRM₁₉₇ Freund's complete adjuvant Tetanus toxoidFreund's incomplete adjuvant Ovalbumin Freund's incomplete adjuvant BSAFreund's incomplete adjuvant KLH Freund's incomplete adjuvant DiptheriaCRM₁₉₇ Freund's incomplete adjuvant Tetanus toxoid

In an alternative embodiment, an immunogenic composition comprising ahapten compound may further comprise a pharmaceutically acceptableexcipient. Non-limiting examples of pharmaceutically acceptableexcipients include sterile water, salt solutions such as saline, sodiumphosphate, sodium chloride, alcohol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycol, gelatine, mannitol, carbohydrates,magnesium stearate, viscous paraffin, fatty acid esters, hydroxy methylcellulose, and buffer. Other suitable excipients may be used by thoseskilled in that art.

V. Eliciting an Immune Response

Another additional aspect of the invention encompasses administering ahapten compound to a subject to elicit an immune response in thesubject. Typically, such an immune response will generate specificantibodies that recognize one or more of (+) methamphetamine,(+)amphetamine, and (+)MDMA. In one embodiment, the specific antibodiesmay recognize two compounds from the group consisting of (+)methamphetamine, (+)amphetamine, and (+)MDMA. In an exemplaryembodiment, the specific antibodies may recognize all three compounds ofthe group consisting of (+) methamphetamine, (+)amphetamine, and(+)MDMA. In another exemplary embodiment, the specific antibodies mayrecognize all three compounds of the group consisting of (+)methamphetamine, (+)amphetamine, and (+)MDMA, and not recognize (−)methamphetamine, (−)amphetamine, and (−)MDMA. In still another exemplaryembodiment, the specific antibodies may recognize all three compounds ofthe group consisting of (+) methamphetamine, (+)amphetamine, and(+)MDMA, and not recognize over the counter medications.

In one embodiment, the elicited immune response may generate antibodiesspecific for a compound administered to a subject. In certainembodiments, a method for generating specific antibodies for a compoundselected from the group consisting of a compound of formula (I), acompound of formula (II), a compound of formula (III), and a compound offormula (IV), may comprise administering the compound to a subject. Inanother embodiment, the elicited immune response may generate antibodiesspecific for more than one hapten compound of the invention administeredto a subject. For instance, a compound of formula (II) and a compound offormula (IV) may be administered to a subject simultaneously in the samecomposition, contemporaneously in separate compositions, orsequentially. Alternatively, more than one compound listed in Table Amay be administered to a subject simultaneously, contemporaneously, orsequentially.

As used herein, subject refers to any vertebrate capable of mounting animmune response. In one embodiment, a subject may be a rodent.Non-limiting examples of rodents include mice and rats. In anotherembodiment, a subject may be a livestock animal. Non-limiting examplesof livestock animals include cows, pigs, sheep, goats, llamas, andpoultry. In still another embodiment, a subject may be a companionanimal. Non-limiting examples of companion animals include dogs, cats,rabbits, and horses. In an additional embodiment, a subject may be aprimate. Non-limiting examples of primates include lemurs, monkeys,apes, and humans. In a further embodiment, a subject may be a non-humanprimate. In an alternative embodiment, a subject may be human.

In each of the above embodiments, the subjects may be subjects addictedto (+) methamphetamine, (+)amphetamine, and (+)MDMA (and/or ecstasy).Alternatively, the subjects may be at risk for addiction to (+)methamphetamine, (+)amphetamine, and (+)MDMA (and/or ecstasy). Inanother alternative, the subjects may be recovering addicts to (+)methamphetamine, (+)amphetamine, and (+)MDMA (and/or ecstasy).

One skilled in the art will appreciate that a hapten compound of theinvention may be administered in a variety of ways to elicit an immuneresponse. (See generally, Herbert and Fristensen (1986) and Poole(1987).) Generally speaking, the method of administration will depend onthe volume of the composition administered, the solubility of thecomposition, and on the speed of the immune response desired. Moreover,the method of administration may be limited by the subject involved.Typically, a method of administration should be chosen that providesincreased antibody titer, affinity, and duration of antibody response.Non-limiting examples of possible administration methods includesubcutaneous administration, intraperitoneal administration, intravenousadministration, intramuscular administration and intradermaladministration. In one embodiment, a composition comprising a haptencompound of the invention may be administered subcutaneously. In anotherembodiment, a composition may be administered intraperitoneally. In yetanother embodiment, a composition may be administered intravenously. Instill another embodiment, a composition may be administeredintramuscularly. In still yet another embodiment, a composition may beadministered intradermally.

The dosage of hapten compound administered will typically vary with thesubject involved. Generally speaking, in formulating a dosage to beadministered, one skilled in the art should consider the weight of thesubject and the method of administration. Moreover, the dosage may bechosen to increase antibody titer, antibody affinity, and/or duration ofantibody response. For instance, a high dosage may lead to higher titerantibodies, but lower affinity antibodies. In one embodiment, the dosageused may be the lowest dosage possible to generate an antibody responsein at least 70% of the subjects involved. In another embodiment, thedosage used may be the lowest dosage possible to generate an antibodyresponse in at least 75%, 80%, 85%, or 90% of the subjects involved. Inyet another embodiment, the dosage used may be the lowest dosagepossible to generate an antibody response in at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% of the subjects involved. In still yetanother embodiment, the dosage used may be the lowest dosage possible togenerate an antibody response in 100% of the subjects involved.Non-limiting examples of specific dosages for various subjects may befound in the examples.

The schedule of administration should also, generally speaking, bechosen to increase antibody titer, antibody affinity, and the durationof immune response. For instance, a subject might be initiallyadministered a hapten compound of the invention, and then receivebooster administrations thereafter. The frequency and number of boosteradministrations can and will vary with the subject involved. Frequentadministrations may increase titer, but not affinity. Alternatively,less frequent administrations may result in increased affinity. Forinstance, in humans, a schedule may include booster administrations forone or more years following the initial administration. Non-limitingexamples of booster schedules may be found in the examples.

VI. Uses for the Specific Antibodies

A further aspect of the invention encompasses both therapeutic andnon-therapeutic uses for the specific antibodies generated using themethods of section V above.

In certain embodiments, the specific antibodies may be used innon-therapeutic assays, such as immunostaining, immunoprecipitation,immunoblotting, immunoaffinity purification, and ELISAs. In oneembodiment, the specific antibodies may be used for immunostaining. Inanother embodiment, the specific antibodies may be used forimmunoprecipitation. In yet another embodiment, the specific antibodiesmay be used for immunoblotting. In still another embodiment, thespecific antibodies may be used for immunoaffinity purification. Instill yet another embodiment, the specific antibodies may be used forELISAs. Protocols for each of the above non-therapeutic uses are wellknown in the art, and may be found, for instance, in Harlow and Lane,Antibodies, Cold Spring Harbor, 1988, Chapters 9-14.

Additionally, the specific antibodies may be used for therapeuticpurposes. Generally speaking, the specific antibodies may be used toantagonize the effects of (+) methamphetamine, (+)amphetamine, and/or(+)MDMA in a subject. In certain embodiments, the subjects may beaddicted to (+) methamphetamine, (+)amphetamine, and/or (+)MDMA. Forinstance, in one embodiment, the specific antibodies may antagonize theeffects of (+) methamphetamine, (+)amphetamine, and/or (+)MDMA in asubject by decreasing the concentration of (+)methamphetamine,(+)amphetamine, or (+)MDMA in the brain of a subject. In anotherembodiment, the specific antibodies may be used to decrease drug-seekingbehavior in a subject. In yet another embodiment, the specificantibodies may be used to decrease self-dosing behavior in a subject.

In each of the above embodiments, the specific antibodies may beadministered passively, actively, or in a combination of passive andactive administration. Active administration typically refers toadministering a hapten compound of the invention to a subject so as thesubject generates antibodies in vivo. Passive administration typicallyrefers to administering at least one specific antibody, generated by asubject or produced via ex vivo methods, into a second subject.

In one embodiment, the invention may encompass a method of treating drugaddiction. The method may comprise eliciting an immune response in adrug-addicted subject by administering a hapten composition to asubject, wherein the immune response decreases the concentration of(+)methamphetamine, (+)amphetamine, or (+)MDMA in the brain of thesubject.

Definitions

To facilitate understanding of the invention, a number of terms aredefined below:

As used herein “(d)” stands for dextrorotatory and (1) stands forlevorotatory, and refers to the direction in which an enantiomer rotatesthe plane of polarized light. Herein, (d) is used interchangeably with(+), and (1) is used interchangeanly with (−).

Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to eight carbon atoms in theprincipal chain and up to 30 carbon atoms or more. They may be straightor branched chain or cyclic and include methyl, ethyl, propyl,isopropyl, butyl, hexyl and the like. Unless otherwise indicated, thealkenyl groups described herein are preferably lower alkenyl containingfrom two to eight carbon atoms in the principal chain and up to 30carbon atoms or more. They may be straight or branched chain or cyclicand include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl,hexenyl, and the like.

Unless otherwise indicated, the alkynyl groups described herein arepreferably lower alkynyl containing from two to eight carbon atoms inthe principal chain and up to 30 carbon atoms or more. They may bestraight or branched chain and include ethynyl, propynyl, butynyl,isobutynyl, hexynyl, and the like.

As used herein, antibody generally means a polypeptide or protein thatrecognizes and can bind to an epitope of an antigen. An antibody, asused herein, may be a complete antibody as understood in the art, i.e.,consisting of two heavy chains and two light chains, or be selected froma group comprising polyclonal antibodies, ascites, Fab fragments, Fab′fragments, monoclonal antibodies, chimeric antibodies, humanizedantibodies, human antibodies, and a peptide comprising a hypervariableand/or framework region of an antibody.

As used herein, antibody affinity refers to the attraction between andantibody and a target epitope. Affinity may be measured by calculating aK_(D) value for a particular antibody and a particular epitope.Typically, affinity can be equated with (1/K_(D)). “K_(D)” as usedherein, refers to disassociation constant. Methods of calculating K_(D)values are well known in the art.

As used herein, antibody titer refers to the concentration of antibodiespresent in the highest dilution of a serum sample at which visibleclumps with an appropriate antigen are formed. Titer may be measuredusing an ELISA or RIA assay, by methods commonly known in the art.

The terms “aryl” or “ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Phenyl andsubstituted phenyl are the more preferred aryl.

The term “conjugate” refers to a substance formed from the joiningtogether of two parts. Representative conjugates in accordance with thepresent invention include those formed by the joining together of asmall molecule and a large molecule, such as a protein. Methamphetamineattached to a carrier protein via a linker is an example of conjugation.

The term “contiguous” is used herein to describe the number of atomsforming a linker. The number of atoms in a linking group or linker isdetermined by counting the contiguous atoms other than hydrogen. In thiscontext, “contiguous” is the number of atoms in a chain within a linkinggroup determined by counting the number of atoms other than hydrogenalong the shortest route between the substructures being connected.

The term “derivative” refers to a chemical compound or molecule madefrom a parent compound by one or more chemical reactions.

The term “hapten” refers to a partial or incomplete antigen. Haptens areprotein-free substances that generally are not capable of stimulatingantibody formation, but may react with antibodies. Amphetamine,methamphetamine, and their derivatives are haptens.

The terms “heterocyclo” or “heterocyclic” as used herein alone or aspart of another group denote optionally substituted, fully saturated orunsaturated, monocyclic or bicyclic, aromatic or nonaromatic groupshaving at least one heteroatom in at least one ring, and preferably 5 or6 atoms in each ring.

The heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfuratoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded tothe remainder of the molecule through a carbon or heteroatom. Exemplaryheterocyclo groups include heteroaromatics such as furyl, thienyl,pyridyl, oxazolyl, pyrrolyl, indolyl, quinolinyl, or isoquinolinyl andthe like. Exemplary substituents include one or more of the followinggroups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protectedhydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen,amido, amino, nitro, cyano, thiol, ketals, acetals, esters and ethers.

The term “heteroaromatic” as used herein alone or as part of anothergroup denotes optionally substituted aromatic groups having at least oneheteroatom in at least one ring, and preferably 5 or 6 atoms in eachring. The heteroaromatic group preferably has 1 or 2 oxygen atoms, 1 or2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may bebonded to the remainder of the molecule through a carbon or heteroatom.Exemplary heteroaromatics include furyl, thienyl, pyridyl, oxazolyl,pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like. Exemplarysubstituents include one or more of the following groups: hydrocarbyl,substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl,acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino,nitro, cyano, thiol, ketals, acetals, esters and ethers.

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl.

As used herein, a “linking group” or “linker” refers to a portion of achemical structure that connects two or more substructures such ashaptens, carriers, immunogens, labels, tracers or other linkers. Alinking group has at least 1 uninterrupted chain of atoms other thanhydrogen (or other monovalent atoms) extending between thesubstructures. The atoms of a linking group and the atoms of a chainwithin a linking group are themselves connected by chemical bonds.Linkers may be straight or branched, saturated or unsaturated,hydrocarbyl or substituted hydrocarbyl chains.

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted with ahetero atom such as nitrogen, oxygen, silicon, phosphorous, boron,sulfur, or a halogen atom. These substituents may include one or more ofthe following groups: halogen, carbocycle, carboxy, aryl, heterocyclo,alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protected hydroxy, keto,acyl, acyloxy, nitro, amino, amido, nitro, cyano, thiol, ketals,acetals, esters and ethers.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES

The following examples illustrate various embodiments of the invention.

Example 1 Protocol for Generation of High Affinity Monoclonal Antibodiesand Fab Fragments that Bind to D-Methamphetamine and Other StimulantDrugs

The haptens are coupled to a bovine serum albumin antigen by using ageneral synthesis procedure (Minh-Tam et al., 1981). This two-step,modified carbodiimide procedure permits a defined number of haptens tobe covalently bound to the protein in a controlled molecularorientation. It also minimizes cross linking of protein molecules andthe unwanted conjugation of the haptens through the free amino group onthe d-methamphetamine haptens. A complimentaryovalbumin-d-methamphetamine hapten was also generated for use inscreening hybridoma products in an enzyme-linked immunosorbent assay(ELISA). This general synthesis procedure has been used in the past togenerate anti-drug antibodies (Owens et al., 1988) and anti-peptideantibodies (Laurenzana et al., 1995).

For the production of monoclonal antibodies, BALB/c mice (n=6-10 perhapten) are immunized with 100 μg of the BSA-d-methamphetamine,emulsified in an equal volume of an adjuvant (e.g., Titer Max, RIBI,Freund's Complete Adjuvant). One month later the animals were boostedwith the same reagents and two weeks later the serum was tested forspecific antibodies using the ovalbumin-d-methamphetamine conjugates inan ELISA. The spleen from the animal with the highest titer ofanti-d-methamphetamine antiserum was used for the first fusion. Theother animals were boosted every three to four weeks to maintain titersof anti-d-methamphetamine in anticipation of future immunizations. Afterfusion of spleen cells from the mice with a myeloma cell line,hybridomas secreting anti-d-methamphetamine antibodies were identifiedusing an ELISA with the appropriate ovalbumin-d-methamphetamineconjugate as described (Laurenzana et al., 1995).

Wells with a positive reaction to d-methamphetamine were subcloned tomonoclonality. For specificity determinations, the antibodies weretested in an ELISA format using a series of ligands. These ligandsinclude (but are not limited to) d- and 1-methamphetamine, d- and1-amphetamine, MDMA, MDA, ephedrine, pseudoephedrine, and otherpotentially cross reacting stimulant-like molecules and endogenousneurotransmitters. Antibodies specific for the d-isomers and having alow K_(D) value (e.g., <1-30 nM) were selected. Although a range ofantibody affinities (as great as 250 nM) has been studied, the objectivewas to have affinity constants for methamphetamine in the range of 1-30nM.

Once an anti-d-methamphetamine secreting hybridoma was chosen, largequantities of the antibody were produced in a hollow fiber bioreactor(Valentine and Owens, 1996). A representative method for the monoclonalantibody purification process is as follows. Monoclonalantibody-containing tissue culture media was combined and concentratedto one-tenth of the original volume using an Amicon spiral cartridgeconcentration system. This technique takes approximately 10 minutes toconcentrate 2 L down to 100-200 ml. The procedure recovers 95% of themonoclonal antibody and removes >95% of the bovine albumin in the media.The concentrated monoclonal antibody was dialyzed against 50 mM MESbuffer (2-(N-Morpholino)-ethanesulfonic acid), pH 6.0 for furtherpurification using a large, glass chromatography column packed with 1 Lof SP-Sepharose Big Bead media (Pharmacia LKB Biotechnology). The samplewas loaded on the column and washed with the MES buffer to removenon-specifically bound proteins. The monoclonal antibody was eluted inone step using 50 mM MES/0.15 M NaCl. This elution also serves toreconcentrate the monoclonal antibody. The purity and concentration ofthe purified anti-d-methamphetamine monoclonal antibody were determinedby SDS-PAGE and spectrophotometry respectively.

Fab fragments of the monoclonal antibody were produced by the papaindigestion method described by Goding (1983) using an mAb:papain ratio of500:1 (w/w). After digestion, the Fab was purified using a HPLC columncontaining Pharmacia Streamline DEAE Sepharose anion exchange media.Purity was checked by SDS-PAGE and the protein concentration wasmeasured with a Coomassie protein assay or spectrophotometrically. Forevery 100 g of monoclonal antibody, one may expect to yield at least55-68 g of Fab fragments. For use in animals, the Fab and monoclonalantibody were dialyzed against PBS, pH 7.4 and concentrated with anAmicon ultrafiltration device to 50-100 mg/ml (depending on the needs ofthe in vivo testing procedure). Both Fab and monoclonal antibody werestored at −80° C. until needed. There was no decrease in bindingactivity or solubility after long-term storage of the monoclonalantibody or Fab.

Example 2 Protocol for the Synthesis Of Hapten Compounds

The methamphetamine-like stimulants that are most often abused aremethamphetamine, amphetamine and MDMA (FIG. 3). Based on review of theliterature on anti-methamphetamine antibodies (e.g., Faraj et al., 1976;Usagawa et al., 1989; Ward et al., 1994) and analysis of the molecularfeatures of the molecules shown in FIG. 3, it is hypothesized thatcoupling of a spacer group (with a carboxylic acid terminus) at the paraor meta position of the aromatic ring structure will offer the bestchance for generating a class-specific antibody. The resultingantibodies are expected to react best with the parent compound, asopposed to metabolites, and would also be less likely to significantlycross react with natural neurotransmitters. If the protein was coupledto the amine groups at the other end of the molecule (which would bemore convenient), this would not generate antibodies that would crossreact with MDMA. The haptens designed for generating antibodies specificfor methamphetamine-like stimulants are illustrated in FIGS. 4A-4C.

A method of using activated ester to couple the hapten to a protein tomake the antibody is shown in FIG. 5. Similar chemistry would apply toall other structures shown in FIGS. 4A-4C.

The synthesis of one of the haptens (hapten 1 in FIG. 4A with X=5 andconnected at the 3-position) is outlined in FIG. 6. The goal is toprepare the (S)-(+)-isomer of 3-(5′-carboxy-pentyloxy)methamphetamine(compound 9 of FIG. 6). To establish the feasibility of the syntheticmethods, the synthesis of (R)-(−)-9 is presented. Those skilled in theart will know that (S)-(+)-9 can be prepared using the same methodstarting with (S)-α-methylbenzylamine.

Thus, to prepare (R)-9,3-methoxyphenylacetone (compound 1 of FIG. 6) wascondensed with (R)-α-methylbenzylamine to give compound 2. Raney nickelreduction of compound 2 followed by separation provided the pure(R,R)-diastereoisomer of compound 3. The N-formyl-protected intermediatecompound 4 was obtained by treating compound 3 with a formic acid-aceticanhydride mixture. O-Demethylation of compound 4 using boron tribromideyielded the phenol compound 5. Alkylation of compound 5 with methyl6-bromohexanoate afforded compound 6. Reduction of compound 6 usingdiborane provided the N—CH₃ intermediate compound 7, which yieldedcompound 8 on reduction using palladium on carbon catalyst in refluxingformic acid. The desired final optically pure hapten compound 9 as thehydrochloride salt was obtained by treating compound 8 with dilutehydrochloric acid.

Example 3 Synthesis of the intermediate compound(R,R)—N-α-Methylbenzyl-3-Methoxyamphetamine Hydrochloride

A solution of 3-methoxyphenylacetone (10 g, 0.061 mol) and(R)-α-methylbenzylamine (7.38 g, 0.061 mol) in 100 mL of dry toluene washeated to reflux in a flask fitted with a Dean-Stark condenser for 20 h.After cooling the reaction mixture, the solvent was removed, and theresidue was dried under vacuum. The residual oil was dissolved inabsolute EtOH (60 mL), and a slurry of EtOH washed Raney nickel wasadded. The resulting mixture was hydrogenated for 96 h at 40 psihydrogen. The catalyst was removed by filtration over a Celite bed, andthe filtrate was treated with HCl gas. Evaporation of the solvent gave awhite solid which was triturated with hot acetone to provide the targetcompound 3 of FIG. 6 as a white solid. An analytical sample was preparedfrom an aliquot removed. The sample recrystallized from MeOH/diethylether had mp 215-218° C.; [a]²¹D (17.85°, c 1.95, MeOH). ¹H NMR (CD₃OD)δ 1.17 (d, 3H), 1.69 (d, 3H), 2.53 (dd, 1H), 3.17 (m, 1H), 3.31 (m, 1H),3.74 (s, 3H), 4.63 (q, 1H), 6.59 (s, 1H), 6.62 (d, 1H), 6.82 (d, 1H),7.21 (t, 1H), 7.54 (m, 5H). Elemental analysis: calcd. for C₁₈H₂₃NO.HCl:C, 70.69; H, 7.91; N, 4.58; Cl, 11.59. Found: C, 70.51; H, 7.99; N,4.53; Cl, 11.65.

Example 4 Synthesis of the intermediate compound(R,R)—N-formyl-N-α-methylbenzyl-3-methoxyamphetamine

To a stirred solution of formic acid (7.5 mL, 0.2 mol) at 0° C. wasadded acetic anhydride (18.9 mL, 0.2 mol) dropwise. After 30 min, theamine compound 3 of FIG. 6 (3.9 g, 13.7 mmol) in a minimum volume offormic acid was added, and the mixture was stirred overnight. Water wascarefully added, and the mixture was neutralized with dilute NH₄OH. Themixture was extracted with CH₂Cl₂, washed with saturated sodium chloridesolution, and dried over NaSO₄. The residue obtained after evaporationwas purified on a silica gel column eluting with a solvent mixture ofhexane/CH₂Cl₂/CH₃OH (5:14:1) to give 3.83 g (94%) of compound 4 of FIG.6 as a white solid.

Example 5 Synthesis of the intermediate compound(R,R)—N-formyl-N-α-methylbenzyl-3-hydroxy amphetamine

To a stirred solution of compound 4 of FIG. 6 (2.85 g, 10 mmol) inCH₂Cl₂ (30 mL) was added a solution of BBr₃ (4.84 g, 20 mmol) in 50 mLof CH₂Cl₂. After stirring overnight, the excess of BBr₃ was quenched bycareful addition of water and the organic fraction separated. Theaqueous layer was further extracted with CH₂Cl₂, and the combined CH₂Cl₂fraction was dried over Na₂SO₄. Evaporation gave 2.01 g (74%) ofcompound 5 of FIG. 6 as a white solid. Further purification on a silicagel column eluting with hexane/CH₂Cl₂/MeOH (4:8:1) gave 1.65 g (61%)pure product. The analytical sample was triturated with ether to givewhite crystals; mp 174-177° C. Elemental analysis: calcd. forC₁₈H₂₁NO₂.1.25H₂O: C, 75.69; H, 7.50; N, 4.91. Found: C, 75.67; H, 7.46;N, 5.00.

Example 6 Synthesis of the intermediate compound(R)-3-(5′-carbomethoxypentyloxy) methamphetamine

To a suspension of hexane washed sodium hydride (216 mg, 4.32 mmol) in 5mL of DMF was added a solution of(R,R)-3-hydroxyphenyl-2-propyl-N-f-ormamido-N-α-methylbenzylamine(compound 5 of FIG. 6) (1.22 g, 4.32 mmol). After stirring for 30 min atroom temperature, a solution of methyl 6-bromohexanoate (1.36 g, 6.48mmol) in DMF (3 mL) was added and stirred overnight at room temperature.The reaction mixture was diluted with H₂O (50 mL) and extracted withmethylene chloride (3×10 mL). The combined organic fraction washed withsaturated sodium chloride solution and dried over Na₂SO₄. After removalof the solvent, the residue was purified on a silica gel column. Elutingwith a solvent mixture (CH₂Cl₂:hexane:MeOH, 4:14:1) to give 1.68 g (95%)of compound 6 of FIG. 6. ¹H NMR (CDCl₃) δ1.28 (dd, 3H), 1.53 (m, 2H),1.58 (dd, 3H), 1.72 (m, 4H), 2.36 (m, 2H), 2.41 (m, 1H), 2.89 (m, 1H),3.25 (m, 1H), 3.41 (t, 2H), 3.68 (s, 3H), 3.82 (q, 2H), 4.58, 6.07 (2 q,1H), 6.17, 6.67 (2 s, 1H), 6.57, 6.40 (2d, 1H), 6.67 (dd, 1H), 7.05 (dd,1H), 7.36 (m, 5H), 8.41, and 8.48 (two s, 1H). The sample was used inthe next step without further characterization.

A solution of the above formamide compound 6 of FIG. 6 (1.63 g) wastreated with BH₃.THF (10 mL) and stirred for 30 min when the excess ofBH₃ was decomposed with MeOH followed by dilute HCl. The reactionmixture was basified with dilute NH₄OH and extracted with methylenechloride (3×25 mL). The organic fraction was dried over Na₂SO₄ andevaporated to dryness. The oily material was dissolved in MeOH (25 mL),and Pd/C (250 mg) was added. The mixture was heated to reflux withformic acid (3 mL in three portions) for an hour. The filtrate, obtainedafter removal of the catalyst, was evaporated and the resulting residuepurified on a silica gel column. Elution with 10% MeOH in methylenechloride gave 0.84 g (70% overall in two steps) of a clear oil compound8. ¹H NMR (CDCl₃) 1.06 (d, 3H), 1.50 (m, 2H), 1.71 (m, 2H), 1.80 (m,2H), 2.33 (t, 2H), 2.41 (s, 3H), 3.67 (s, 3H), 3.95 (t, 2H), 6.75 (m,3H), 7.19 (m, 1H). The sample was converted to HCl salt; mp 53-57° C.Elemental analysis: calcd. for C₁₇H₂₇NO₃.HCl.0.75H₂O: C, 59.50; H, 8.50;N, 4.10. Found: C, 59.65; H, 8.45; N, 4.21.

Example 7 Synthesis of intermediate (R)-3-(5′-carboxypentyloxy)methamphetamine hydrochloride

A solution of compound 8 of FIG. 6 (400 mg, 1.15 mmol) in dilutehydrochloric acid (6N, 5 mL) was heated to reflux for 4 h. The reactionwas evaporated to dryness, and the residue was crystallized fromMeOH/ether to give 215 mg (59%) of an off-white crystalline material: mp73-77° C. ¹H NMR (CD₃OD) 1.25 (d, 3H), 1.34 (m, 2H), 1.40 (m, 2H), 1.67(m, 2H), 2.65 (t, 2H), 2.72 (s, 3H), 4.22 (m, 2H), 6.73 (m, 3), 7.13 (s,1H). Elemental Analysis: calcd. for C₁₆H₂₅NO₃.HCl.0.25H₂O: C, 59.99; H,8.34; N, 4.37; Cl, 11.07. Found: C, 60.09; H, 8.33; N, 4.37; Cl, 11.13.

Example 8 Effect of Hapten Design on Antibody Specificity forD-Methamphetamine Like Drugs

In these experiments, rabbit antiserum was generated against two uniqued-methamphetamine like haptens. Each hapten included the basic chemicalstructure of d-methamphetamine, along with a new chemical linker groupattached at the para (para-O,6 hapten) or meta (meta-O,6 hapten)positions of the aromatic ring structure (see Table 3 herein). Thedistal end of this linker group had a carboxy terminus for use informing a peptide bond with protein antigens. After synthesis of ahapten-bovine serum albumin conjugate, this antigen was used forimmunizing two rabbits. The first immunization for each rabbit was with200 μg of either para-O,6 antigen or meta-O,6 antigen in Freund'scomplete adjuvant. The first booster immunization was with 100 μg ofantigen in Freund's incomplete adjuvant. Seven to ten days later eachanimal was bled and the serum was collected for testing.

After titering each antiserum for selection of an appropriate serumdilution for radioimmunoassay, the relative cross-reactivity of eachantiserum was determined. In this assay, a constant dilution ofantiserum and a constant amount of [³H]-methamphetamine was added toeach test tube. Next, increasing amounts of either d-amphetamine ord-methamphetamine were added to separate tubes. After an overnightincubation at 4-8° C., the antibody bound [³H]-methamphetamine wasseparated from the free [³H]-methamphetamine using a goat anti-rabbitsecond antibody. The antibody precipitate in each tube was thentransferred to a scintillation vial and the amount of radioactivity ineach tube was determined by liquid scintillation spectrometry. For eachof the test drugs (either d-amphetamine or d-methamphetamine), the ED₅₀value for inhibition of [³H]-methamphetamine binding to each antiserumwas determined using a sigmoidal (logistic) fit to the percentage of[³H]-methamphetamine binding versus log ligand dose.

Results from these studies show that the antiserum generated from thepara-O,6 hapten (right two dose-response curves, FIG. 7) issignificantly more specific for d-methamphetamine (ED₅₀=427 nM) than itis for d-amphetamine (ED₅₀=5157 nM). Indeed the relative crossreactivity for d-amphetamine is only 8.3% (427 nM/5157 nM×100%) of thevalue for d-methamphetamine. Thus, while this hapten might be useful indeveloping a highly specific assay for detection of d-methamphetamine,it would not be useful in generating a monoclonal antibody-basedmedication with high affinity and broad recognition for d-amphetaminelike drugs.

In contrast, results from the radioimmunoassay analysis of the meta-O,6antiserum (left two dose-response curves, FIG. 7) showed d-amphetamine(ED₅₀=47 nM) cross-reactivity is 59.6% (28 nM/47 nM×100%) of the valuefor d-methamphetamine (ED₅₀=28 nM). In these studies the meta-O,6 haptenalso generated higher affinity antiserum than the para-O,6 hapten, asdetermined from the significantly lower ED₅₀ values for bothd-amphetamine and d-methamphetamine. As an object of this invention isto generate a widely cross-reacting antiserum for d-amphetamine-likedrugs, these data reflect the importance of the hapten design onantibody specificity.

Example 9 Comparison of Protocols for Active and Passive Immunization asTreatments for D-Methamphetamine Addiction

A series of male Sprague-Dawley rats are immunized with ad-methamphetamine-like hapten until high titers are achieved, or treatedwith anti-d-methamphetamine mAb. The rats are then repeatedly challengedwith i.v. d-methamphetamine over several weeks. The ability of theantibodies to antagonize drug effects over an extended time period isassessed by behavioral measurements of response and usingd-methamphetamine dose-response curves with dosing schedules that aredesigned to simulate repeated binge use of the drug. The rats for all ofthese studies are purchased with indwelling jugular venous catheters fori.v. administration of d-methamphetamine and anti-d-methamphetamine mAb.

For active immunization, one group of rats (n=6 for all groups) isimmunized over a six week period prior to the start of the studies. Anexample immunization plan is 100 μg of the BSA-d-methamphetamine,emulsified in an equal volume of Titer-Max as adjuvant, followed atweeks 3 and 6 by a booster immunization. Ten days after the last boost,the anti-d-methamphetamine serum titers are checked in an ELISA. If thetiters are elevated, behavioral testing begins on day 10-14 after thelast boost.

For passive immunization, another group of rats is treated with 400 mgdose of monoclonal antibody the day before the start of the study. Thisdose of anti-d-methamphetamine monoclonal antibody (400 mg) should havethe capacity to bind up to 2.1 mg/kg of d-methamphetamine on day 1 ofthe behavioral experiments, and up to 0.52 mg/kg of d-methamphetamine onday 16 (the final day of testing). A 2.1 mg/kg dose of d-methamphetaminein the rat would be about equivalent to a 150 mg binge use ofd-methamphetamine in an average size human (i.e., about 150 lbs). Thecalculation of the d-methamphetamine (M.W. 149 g/mol) mol-eq dose of IgGassumes a 350 g rat, two IgG binding sites, a mass of 150,000 kDa, an invivo first-order monoexponential loss of the IgG, and an IgG t_(1/2) of8.1 days (Bazin-Redureau et al., 1997).

The effectiveness of each therapy is measured by accessing thecumulative behavioral effects after administration of a range ofd-methamphetamine doses over a 3 hr time period. This d-methamphetaminedosing strategy is used to simulate binge drug use and an addict'sattempt to surmount the blocking effects of the antagonist byself-administration of progressively higher doses. The i.v. doses of0.1, 0.3 and 1.0 mg/kg are administered at 0, 1.5 hrs and 3.0 hrs,respectively. This simulated binge dosing is repeated every 4 days (day1, 4, 8, 12 and 16) for up to 16 days.

The EthoVision system, which has video tracking and digitized motionanalysis, is used for continuous behavioral monitoring.d-methamphetamine-induced locomotor activity, e.g., distance traveled,percentage of the time spent moving, and animal rearing, are measuredover a 6 hr period. From each day of behavioral experiments, the time tomaximum effects after each dose of d-methamphetamine, the maximumeffect, the area under the behavioral effect curve from the time ofdosing to the end of each type of behavioral effect, and the duration ofeffects are calculated. The end of each behavioral effect is based on astatistical analysis of the average baseline response prior to drugadministration. For instance, the point at which the animals' responsehas returned to 1+S.D. of the mean pre-drug response for two consecutive2 min intervals. The data are analyzed by a two-way (dose ofd-methamphetamine and time) repeated measures ANOVA, followed by aStudent-Newman-Keuls post hoc test. The results are consideredsignificant at P<0.05.

Example 10 Pharmacodynamic Mechanisms of Monoclonal Antibody-BasedAntagonism of High Dose (+)-Methamphetamine in Rats

This example demonstrates that anti-(+)-methamphetamine monoclonalantibodies antagonize (+)-methamphetamine-induced locomotor effects byaltering brain distribution of (+)-methamphetamine.

Two (+)-methamphetamine-like haptens with either a six- or four-carbonspacer group were used for antibody production. The complete synthesisof the (+)-P6-METH hapten (S-(+)-4-(5-carboxypentyl) methamphetamineHCl) was previously described (Byrnes-Blake et al., 2001). The(+)-P4-METH hapten (S-(+)-4-(3-carboxypropyl) methamphetamine HCl) wassynthesized in a similar fashion. Both haptens were conjugated (Davisand Preston, 1981) to bovine serum albumin (BSA) for use as an antigen.

Female BALB/c mice (Charles River Laboratories, Wilmington, Mass.) wereimmunized with 100 μg of the hapten-conjugates emulsified 1:1 (v/v) withTiterMax adjuvant and boosted every 4 weeks with 50 ug of the antigen.Initial immunization and subsequent boosts were administered in two 40ul subcutaneous injections. Blood samples were taken periodically bytail bleed to measure anti-(+)-methamphetamine antibody titer by anELISA utilizing hapten-ovalbumin conjugates. Mice with the highestanti-(+)-methamphetamine serum titer was chosen for mAb production.Standard hybridoma technology was utilized for the production of themAb. Hybridoma cell lines were screened for anti-(+)-methamphetamineantibody production by ELISA.

A low-affinity mAb (mAb 6H8; K_(D)=250 nM) was generated fromimmunization with the (+)-P4-METH-BSA conjugate, and a higher-affinitymAb (mAb 6H4; K_(D)=11 nM) was generated from immunization with the(+)-P6-METH-BSA conjugate. Both antibodies were IgG₁ with a κ lightchain.

The mAbs were highly specific for (+)-methamphetamine, having <0.1%cross-reactivity with most compounds tested. The one exception was thedrug of abuse MDMA or “ecstasy” to which mAb 6H4 bound with a slightlyhigher relative affinity than (+)-methamphetamine (9 nM vs. 11 nM) (FIG.8). Both mAbs were also stereospecific, having an approximately 50-200times higher relative affinity for (+)-methamphetamine and(+)-amphetamine than for the minus forms of these drugs. In addition tothe compounds shown in FIG. 8, there was no significant cross-reactivitywith (+)- and (−)-MDA, (+)-norpseudoephedrine, L-phenylephrine,(+)-phenylpropanolamine, β-phenylethylamine and tyramine.

The day before being administered to the animals, the mAbs wereultracentrifuged at 100,000 g for 90 min at 4° C. and at 3,300 g for 20min. This step was used to eliminate large-molecular-weight antibodycomplexes that can be highly antigenic. The mAb formulations were warmedto 37° C. before i.v. administration to the animals.

Locomotor Activity in the Rat Model

Locomotor activity was used as a measure of (+)-methamphetamine'seffects because 0.3-3.0-mg/kg doses of (+)-methamphetamine produceddose-dependent and reproducible increases in both distance traveled andrearing (Riviere et al., 1999). Higher doses were not used becausepreliminary studies showed i.v. doses ≧5.2 mg/kg led to self-mutilation,and 10-mg/kg doses were sometimes lethal. The time of mAb treatment(t=30 min) was chosen because (+)-amphetamine formation is near maximum,(+)-methamphetamine distribution to the peripheral tissues is virtuallycomplete (Riviere et al., 1999, 2000), and the drug-induced locomotoreffects are profound at this time point.

Comparison of the Reversal of (+)-Methamphetamine-induced LocomotorActivity by a Low- and High-affinity Anti-(+)-Methamphetamine mAb

To help elucidate the role of antibody affinity as a determinant oftherapeutic efficacy, the low-affinity mAb (mAb 6H8) and thehigh-affinity mAb (mAb 6H4) were compared for their ability to reversethe locomotor activity following a 1-mg/kg i.v. (+)-methamphetaminedose. The high-affinity mAb more effectively antagonized both distancetraveled and rearing than the low-affinity mAb (FIG. 9).

Effect of the Hiqh-affinity Anti-(+)-Methamphetamine mAb on(+)-Methamphetamine-Induced Locomotor Activity

These results show the ability of a fixed mAb dose to antagonize theeffects of (+)-methamphetamine at three different doses. Thetime-dependent pattern of time spent moving was very similar to thetime-dependent pattern of distance traveled, but it appeared to be aless sensitive measure. Therefore, the results for this parameter werenot reported. FIG. 10 shows the time course of both distance traveledand rearing events after (+)-methamphetamine administration both withoutand with mAb treatment. FIG. 11 shows a summary of the total distancetraveled and rearing events during the entire experimental period foreach dosing group.

For both the 0.3 and 1.0 mg/kg (+)-methamphetamine doses, thehigh-affinity mAb substantially reduced the locomotor activities(distance traveled and rearing events) from baseline (+)-methamphetamineactivities by >60% and >70%, respectively (all p<0.05). However, therewas a significant increase in both distance traveled and rearingbehavior when animals received 3.0 mg/kg (+)-methamphetamine followed bythe mAb (p<0.05; FIG. 11).

At the end of the experimental protocol, animals that received the1.0-mg/kg doses of (+)-methamphetamine also received a second salinetreatment followed by buffer. The saline-induced behavior was notsignificantly different from that obtained at the start of the study(p<0.05).

In addition to assessing mAb-induced changes in total distance traveledand the number of rearing events, changes in the duration of(+)-methamphetamine's action were also evaluated to measure the effectsof mAb6H4. The duration of (+)-methamphetamine-induced locomotor effectsfollowing treatment (treatment=buffer vs. mAb) was approximately 1 hcompared with 6 min for the 0.3-mg/kg dose and 2 h compared with 32 minfor the 1.0-mg/kg dose. When the mAb was administered to the 3.0-mg/kggroup, however, the duration of drug action increased from 4 to 6 h(FIG. 10).

(+)-Methamphetamine and (+)-Amphetamine Pharmacokinetic Profile After(+)-Methamphetamine Administration

The disposition of (+)-methamphetamine and its active metabolite,(+)-amphetamine, after a 1.0-mg/kg i.v. (+)-methamphetamine dose weresimilar to those of Riviere et al. (2000). The concentration-versus-timeprofiles of (+)-methamphetamine in serum and brain were best describedby a two-compartment model with 1/y² weighting. The distributionhalf-life of (+)-methamphetamine was 1.7 min in serum and 26 min forbrain. In both serum and brain, the highest (+)-methamphetamineconcentrations were achieved at the earliest measured time point (1 min;FIG. 12), followed by a biexponential decline. The metabolite(+)-amphetamine achieved apparent maximum concentrations in serum andbrain at about 30 min (FIG. 12). Table 1 summarizes the pharmacokineticvalues.

Effect of High-affinity Anti-(+)-Methamphetamine mAb on thePharmacokinetic Profiles of (+)-Methamphetamine and (+)-Amphetamine

Administration of the high-affinity mAb (mAb6H4) at t=30 min after(+)-methamphetamine administration led to a substantial change in thedrug's disposition. Concentrations of (+)-methamphetamine in serum weresignificantly higher, corresponding to lower concentrations in brain(FIG. 13). The AUC_(38 min-1.5 h) value for the serum(+)-methamphetamine concentration-versus-time profile showed a >9000%increase, whereas the AUC_(38 min-1.5 h) for brain showed a >70%decrease. The t_(1/2λZ) values in the mAb-treated animals were notdetermined, as the elimination phase of the concentration-time profilecould not be fully characterized within the 4.5-h experiment. The(+)-methamphetamine AUC_(brain)-to-AUC_(serum) ratio was greatlydecreased due to the large increase in serum concentrations (Table 1).The mAb had a significant effect on serum and brain (+)-amphetamineconcentrations at some of the time points, but the effect was not aslarge as that seen with (+)-methamphetamine (FIG. 14). Because of themAb's differential effects on (+)-methamphetamine and its metabolite(+)-amphetamine, there were major changes in the molar ratio ofAUC_(AMP) to AUC_(METH) in both serum and brain (Table 1).

TABLE 1 Parmacokinetic Parameters Of (+)-METH And Its Metabolite (+)-AMPAfter A 1-mg/kg i.v. (+)-METH Dose^(a) Molar Ratio of T_(1/2λz)AUC_(38 min-1.5 h) (+)-AMP to (+)- Control Treated Control TreatedAUC_(brain) AUC_(serum) METH AUC^(b) Tissue Drug h ng · h/ml or ng · h/gControl Treated Control Treated Serum (+)-METH 1.08 NC 123 12,266 1 10.49 0.01 Serum (+)-AMP 1.8 NC 55 73 1 1 Brain (+)-METH 0.95 NC 1182 2469.6 0.02 0.49 1.26 Brain (+)-AMP 1.5 NC 530 284 9.6 3.4 ^(a)Data areshown from animals both without treatment (control) and with treatment(1-mol equivalent dose of mAb). All parameters were calculated bymodel-independent analysis. NC, not calculated due to inadequatesampling during the terminal phase. ^(b)The molar ration of (+)-AMP to(+)-METH AUC was calculated by dividing the nmol · h/g or nmol · h/mlAUC values.

Discussion for Example 10

The overall goal of these studies was to determine the mechanismsassociated with anti-(+)-methamphetamine mAb-based antagonism of(+)-methamphetamine-induced locomotor effects in a rat overdose model.The influence of mAb affinity on therapeutic success was first examinedbecause there was no previous studies addressing this important issue.Two mAbs were developed for these studies. Both mAbs were of the sameisotype and light chain and were highly specific for(+)-methamphetamine. They differed in only one important aspect: a25-fold difference in K_(D) values.

The higher-affinity mAb was two to three times more effective than thelower-affinity mAb at reducing (+)-methamphetamine-induced distancetraveled and rearing events (FIG. 10). However, even the higher-affinitymAb6H4 did not achieve the maximum possible antagonism against theeffects of (+)-methamphetamine, and experimental evidence suggests thatanother 10- to 25-fold increase in mAb affinity would be needed tosignificantly improve the therapy further. The hypothesis that a higheraffinity mAb (e.g., K_(D)=1 nM) would offer substantial improvements issupported by other studies showing that an anti-phencyclidine mAb with aK_(D) of 1.8 nM can completely reverse phencyclidine's locomotor effects(Hardin et al., 1998). A single dose of that antibody can reduce brainconcentrations of phencyclidine for at least one month (Proksch et al.,2000). However, it should be noted that the (+)-methamphetamine ratmodel is complicated by the presence of a pharmacologically activemetabolite, (+)-amphetamine, with which mAb6H8 and mAb6H4 do notcross-react. A mAb with increased cross-reactivity for (+)-amphetamine(or a cocktail of an anti-(+)-methamphetamine and ananti-(+)-amphetamine mAb) may improve the effectiveness of the therapy.

Due to its relatively superior effectiveness, the high-affinity mAb wasused for all subsequent behavioral and pharmacokinetic studies. A fixeddose of the high-affinity mAb (equimolar to the (+)-methamphetamine bodyburden at 30 min after a 1-mg/kg (+)-methamphetamine dose) effectivelyantagonized (+)-methamphetamine-induced effects when the(+)-methamphetamine dose was equal to or less than the mAb dose (FIGS.10 and 11). The mAb also significantly decreased the drug's duration ofaction at both (+)-methamphetamine doses (FIG. 10). However, when thedrug dose was greater than the mAb binding capacity (i.e., at the3.0-mg/kg), locomotor activity was increased after mAb administrationcompared with that seen after (+)-methamphetamine administrationfollowed by buffer.

Several possible explanations exist for these apparent complex changesin behavior at the high (+)-methamphetamine-to-mAb ratio. First, thelocomotor activity could have maximized at doses somewhere between 1 and3 mg/kg. This is described as a so-called inverted-U-shapeddose-response curve. If this were the case, mAb administered at a doseequimolar to a 1-mg/kg (+)-methamphetamine dose could have neutralizedpart of the drug dose, thus shifting the dose-response curve back to thepoint of an apparent increase in locomotor activity. This hypothesis wastested by quantitating locomotor activity in rats after administering a1.8-mg/kg (+)-methamphetamine dose (a half log dose between 1- and3-mg/kg). The 1.8-mg/kg dose produced locomotor effects that were aboutequal to the effects of the 1- and 3-mg/kg (+)-methamphetamine doses(data not shown). Thus, the increased activity following mAb treatmentin the 3.0-mg/kg-dose group could not be explained by a simple shift tothe left in the (+)-methamphetamine dose-response curve.

A possible pharmacodynamic reason for the increase in the behavior isdescribed as follows. It is quite likely that (+)-methamphetamine'smultiple mechanisms of action in the brain and peripheral sites havedifferent concentration-response relationships and are more or lesssusceptible to the neutralizing, beneficial effects of the mAb. Thus,when the mAb is present in limited amounts relative to the amount ofdrug, the mAb would presumably have the greatest effects on the mostaccessible and lowest-affinity effector sites. It is also possible thatthe mAb could preferentially neutralize the behaviorally suppressiveeffects, like stereotyped behavior, while allowing the stimulatoryeffects to predominate.

There are also pharmacokinetic and immunological explanations for theincrease in total locomotor activity. Firstly, the mAb appeared to havesubstantially slowed (+)-methamphetamine's entry into the CNS throughhigh-affinity binding in serum (FIG. 13). This would have led to adecreased amount of (+)-methamphetamine reaching the CNS but prolongedavailability of the drug. Secondly, significant amounts of the activemetabolite, (+)-amphetamine, may have accumulated because(+)-amphetamine has a longer half-life than (+)-methamphetamine in rats(Riviere et al., 1999, 2000; Cho et al., 2001; Table 1). In addition,(+)-amphetamine does not significantly cross-react with the mAb.Thirdly, the association and dissociation of (+)-methamphetamine withthe mAb and its relationship to the influx and efflux of the drug in theCNS could also be factors. In an attempt to address this point, thebehavioral effects following a 3-mg/kg intraperitoneal (i.p.)(+)-methamphetamine dose were compared with those of the 3-mg/kg i.v.dose. The i.p. route was chosen because it provides a model of a slowerdrug input into the brain and would produce greater (+)-amphetamineconcentrations due to liver first-pass metabolism. The data showed thatthe i.p. route of administration resulted in significantly increasedeffects compared with those of the i.v. route. These findings suggestthat the rate of drug entry in the CNS and the increased (+)-amphetamineconcentrations are important factors in (+)-methamphetamine-inducedlocomotor activity and the effectiveness of the mAb.

Pharmacokinetic studies were conducted to help determine some of themechanisms for the behavioral effects. These studies were carried outwith a 1:1 molar ratio of (+)-methamphetamine to mAb. Serum was chosenbecause (+)-methamphetamine is transported to its sites of action viathe bloodstream and mAbs are confined mainly to the serum volume. Thebrain was chosen for study because it is the major site of actioncontributing to (+)-methamphetamine's locomotor effects.

Immediately after the administration of mAb6H4, serum(+)-methamphetamine concentrations dramatically increased and remainedhigh throughout the duration of the experiment (FIG. 13). Theconcentrations were measured for only 4.5 h so that better understandingof the pharmacokinetic processes associated with thepharmacological-effect period of (+)-methamphetamine (FIG. 10) can beobtained. The mAb also produced immediate decreases in brain(+)-methamphetamine concentrations which were sustained for at least 3h. This immediate reduction in brain concentrations was totallyconsistent with the immediate reversal of (+)-methamphetamine-inducedlocomotor effects at the 1-mg/kg dose (FIG. 10). However, by 4.5 h, thebrain concentration appeared to rebound and was then not significantlydifferent from that of the control animals. By this point, however, the(+)-methamphetamine concentration in control and treatment animalsappeared to be below the threshold concentration leading to locomotoractivity. These pharmacokinetic changes for (+)-methamphetamine areconsistent with a similar dramatic decrease (and rebound) inphencyclidine brain concentrations after anti-phencyclidine Fab fragmenttreatment in rats (Valentine and Owens, 1996). It is believed that thesechanges resulted from the rapid mAb-induced redistribution of drug fromthe brain, followed by an increase in drug concentrations due to aslower redistribution from other tissues.

The effect of the mAb on (+)-amphetamine concentrations was small,compared to its effect on (+)-methamphetamine concentrations. This isnot surprising because the mAb had little cross-reactivity with(+)-amphetamine in vitro. The small mAb-induced increase in serumconcentrations of (+)-amphetamine may result from increased amounts of(+)-methamphetamine in the serum available for metabolism. Thishypothesis can be proved by knowing the free concentrations and serumclearance of (+)-methamphetamine. The small decrease in (+)-amphetamineconcentrations in brain and the dramatic decrease of (+)-methamphetamineconcentrations in brain suggest the possibility of brain conversion of(+)-methamphetamine to (+)-amphetamine. However, data are insufficientto fully support this conclusion.

These data indicate that the 70% decrease in brain (+)-methamphetamineAUC is a direct cause of the 70% decrease in behavioral effectsfollowing mAb treatment at the 1-mg/kg dose. In addition,(+)-amphetamine formation does not play a significant role in(+)-methamphetamine's pharmacological effects at this dose. This wouldnot be the case at the 3-mg/kg dose because brain (+)-amphetamineconcentrations would be approximately threefold greater at all timepoints. The ratio of the (+)-amphetamine AUC to the (+)-methamphetamineAUC in serum and brain of rats is about 49% (Riviere et al., 2000).However, the amounts of (+)-amphetamine in humans appear to besubstantially lower (Cook et al., 1993). Therefore, the prediction ofbeneficial effects in humans based on rat data is somewhat hindered bythe very high (+)-amphetamine-to-(+)-methamphetamine ratio in the rat.

In conclusion, the effects of mAb therapy for (+)-methamphetamine aredependent on mAb affinity, dose and specificity. Monoclonal antibodieswith improved affinity for (+)-methamphetamine, and possibly increasedcross-reactivity with (+)-amphetamine, could offer improvements in theeffectiveness of the therapy.

Animal Protocols for Example 10

Male Sprague-Dawley rats were obtained from Hilltop Laboratories(Scottsdale, Pa.) with an indwelling jugular venous catheter (Silasticmedical-grade tubing, 0.020-inch inner diameter and 0.037-inch outerdiameter; Dow Corning Corporation, Midland, Mich.). Catheter patency wasmaintained with a saline and 25 U of heparin flushes every othermorning. The rats were housed separately, and their weight wasmaintained between 270-300 g throughout the experiment. All animalexperiments were conducted with the approval of the Institutional AnimalCare and Use Committee of the University of Arkansas for MedicalSciences and were in accordance with the Guide for the Care and Use ofLaboratory Animals adopted and promulgated by the National Institutes ofHealth.

Protocol for (+)-Methamphetamine Locomotor Activity Studies of Example10

The parameters of distance traveled (in centimeters or meters), numberof rearing events, and time spent moving (in seconds) were individuallyquantified for each animal. The general protocol was previouslydescribed (Hardin et al., 1998; Riviere et al., 1999). Briefly, animalswere placed in polyethylene chambers that contained a bedding ofdark-gray clay. Animal behavior was videotaped, and the video imageswere digitized and quantitated in 4-min intervals by EthoVision software(Noldus Information Technology Inc., Sterling, Va.). The duration ofdrug action was calculated for each parameter starting at 30 min (timeof treatment) and until the locomotor activity returned to baseline.Locomotor activity was determined to have returned to baseline levelswhen two consecutive 4-min intervals were less than or equal to themean+1 S.D. of the 30-min behavioral baseline observed just before drugadministration.

Rats were habituated to the behavioral monitoring chambers before thestart of the experimental protocol. This was accomplished by placing therats in the chambers for a minimum of 3 h per day for 4-6 consecutivedays. After the habituation phase, the rats were then randomly dividedinto three (+)-methamphetamine-dosing groups (n=6 rats per group). Therats in each group were dosed every three days for a total of 10 days.All saline and (+)-methamphetamine injections were administered via thejugular catheter as a 15-sec i.v. infusion.

On day 1, all groups received saline followed at 30 min by mAb buffer toobtain baseline (non-drug-induced) behavior. Then on days 4 and 7, theyreceived a pretreatment dose of either 0.3, 1.0, or 3.0 mg/kg(+)-methamphetamine followed at 30 min by buffer. Two(+)-methamphetamine sessions were conducted because preliminary studiesshowed that, on average, the rats had a lower(+)-methamphetamine-induced locomotor response to the first i.v.injection of the drug than to the second and subsequent injections (datanot shown). Thus, the second pretreatment drug dose was used todetermine the (+)-methamphetamine-induced locomotor activity baseline.

The third and final dose of 0.3, 1.0, or 3.0 mg/kg (+)-methamphetaminewas administered on the last day of the protocol. This dose was followedat t=30 min by i.v. injection of the high-affinityanti-(+)-methamphetamine mAb (mAb6H4; K_(D)=11 nM). The 30-min timepoint was chosen for treatment because previous studies showed thatlocomotor activity and active metabolite concentrations((+)-amphetamine) in tissues are near maximum at 30 min (Riviere et al.,1999, 2000). The amount of mAb administered was 367 mg/kg, which isequimolar (assuming two binding sites per IgG molecule) to the amount of(+)-methamphetamine left in the body at t=30 min. The amount of drugremaining was determined from the serum pharmacokinetic data fromRiviere et al. (1999) and by the equation: body burden=dose x e^(−kt)(Rowland and Tozer, 1995). This simple monoexponential elimination phaseequation could be used to calculate the body burden at 30 min becausethe distribution phase for (+)-methamphetamine in serum is extremelyshort (t_(1/2dist)=9 min).

All control buffer and mAb solutions were administered via the jugularvenous catheter in an 8-ml volume at 2 ml/min. Three days after the endof the experimental protocol for the 1-mg/kg group, locomotor activityfrom a second saline administration was monitored to determine whetherany changes had occurred in baseline activity.

In another study, the therapeutic effectiveness of the low-affinity mAb(mAb6H8; K_(D)=250 nM) and that of the high-affinity mAb were directlycompared. Data from the 1-mg/kg group in the experiment described in theprevious paragraphs (with mAb6H4) were compared with data from a groupof rats (n=6) administered 1 mg/kg (+)-methamphetamine and treated att=30 min with mAb6H8. All other aspects of the protocol were as justdescribed.

Protocol for (+)-Methamphetamine and (+)-Amphetamine PharmacokineticStudies of Example 10

Before each pharmacokinetic experiment, a 1-mg/kg (+)-methamphetaminedose plus a 333-μCi/kg ³H-(+)-methamphetamine dose was prepared insterile saline. This allowed the administration of approximately 100 μCiper rat by injection of 1 μl/g of rat body weight.

Male Sprague-Dawley rats were randomly placed into two groups. The firstgroup (n=3 per time point for all pharmacokinetics studies) was thecontrol group that did not receive antibody. Rats in this group wereadministered a 15-sec i.v. injection of the(+)-methamphetamine/[³H]-(+)-methamphetamine solution via the jugularvenous catheter and were then placed in metabolism cages (Nalge NuncInternational, Rochester, N.Y.). At various predetermined times afterinjection (1, 5, 15, 29, and 38 min; 1, 2, 3, and 4.5 h), the rats weresacrificed. At the early time points (1 and 5 min), the rats wereanesthetized before drug injection so that an immediate laparotomy couldbe performed to obtain blood from the inferior vena cava, anddecapitation could take place at the correct time. At later time points(15 min onward), rats were anesthetized 5 min before the desired time ofsacrifice (decapitation) to allow time for obtaining sufficient depth ofanesthesia and for the laparotomy and blood collection to take place.Ethyl ether was used for anesthesia so that hemodynamic stability couldbe maintained before animal sacrifice. Immediately after bloodcollection, the rats were decapitated and the brain was removed. Thebrain was rinsed with saline, weighed, and placed in liquid nitrogenwithin 3 min of decapitation. Hematocrit values were obtained for eachanimal. The blood was allowed to clot and serum was obtained aftercentrifugation. The serum and brain tissues were stored at −80° C. untilanalyzed.

The second group was the mAb-treatment group. All aspects of theexperiment were same as those described for the control group, with thefollowing exception. At t=30 min, each rat was administered 367 mg/kg ofthe mAb (equimolar to the body burden of (+)-METH in the rat at 30 min).The mAb was given via the jugular venous catheter in an 8-ml volume at 2ml/min. Because the experimental protocol was the same up until the mAbwas administered, the time points before 30 min were not repeated inthis group.

Analysis of Drug Concentrations of Example 10

(+)-methamphetamine and (+)-amphetamine were extracted from serum andbrain with the use of a solid-phase extraction procedure. For serumanalysis, 300 μl of guanidine HCl was added to 200-μl of each serumsample to denature the proteins. The samples were vortexed and placed ona gentle shaker for 30 min. Then, 120 μl of a solution containing0.025-mg/ml (+)-methamphetamine/(+)-amphetamine internal standard wereadded, and each sample mixture was placed directly onsolvent-conditioned Oasis HLB extraction cartridges (1 ml, 30 mg; WatersCorporation, Milford, Mass.). After sample application, the cartridgeswere centrifuged at 230 g for 1 min, washed with 1 ml of water, and thencentrifuged. For elution of both (+)-methamphetamine and(+)-amphetamine, the cartridges were transferred to siliconized testtubes, 1 ml of methanol was added, and 1 min of centrifugation followed.Then, 1 ml of methanol:acetic acid (98:2) was added, followed bycentrifugation.

To determine (+)-methamphetamine and (+)-amphetamine concentrations inthe brain, tissues were homogenized for 30 sec in 5× (v/w; mug) ice-coldwater with a tissue homogenizer (Tekmar Company, Cincinnati, Ohio). A200-μl aliquot was then added to 300 μl of 8 M guanidine HCl. Themixture was vortexed and gently shaked for 30 min. Then, 500 μl ofwater, 120 μl of the (+)-methamphetamine/(+)-amphetamine internalstandard, and 300 μl of a 10% ZnSO₄ solution (to precipitate proteins)were added. The mixture was vortexed, placed on ice for 5 min, andcentrifuged at 12,500 g for 5 min.

Supernatants from the brain samples were applied to conditionedextraction cartridges and then centrifuged at 600 g for 4 min. The brainprecipitates left over from the 12,500 g centrifugation were resuspendedin 500 μl of water and centrifuged at 12,500 g for 3 min. Thesupernatants were then added to their respective extraction columns andcentrifuged at 600 g for 4 min. This was followed by a 1-ml water wash,with another 4 min centrifugation at 600 g. Finally, the cartridges wereplaced in siliconized test tubes for sample collection. The elutionprocess was as described for the serum samples.

After elution, the samples were taken to dryness over 3 h in a vacuumcentrifuge (Savant Instruments, Inc., Farmingdale, N.Y.) with no heat orcryopumping. They were resuspended in 120 μl of 7% acetonitrile and 93%water (the HPLC starting conditions). A Waters Corporation HPLC systemconsisting of a pump controller, autoinjector, UV detector, Millenniumsoftware, and a Symmetry Shield RP18 (3.5 μm, 4.5×75 mm) column was usedto separate (+)-methamphetamine and (+)-amphetamine for quantitation.The mobile phase was 7% acetonitrile and 93% water with 0.1%trifluoroacetic acid. Fractions (10 sec) were collected, and the³H-(+)-methamphetamine and ³H-(+)-amphetamine containing fractions werequantified by liquid scintillation spectrometry. The serum and braindrug concentrations were determined from the ratio of unlabeled(+)-methamphetamine or (+)-amphetamine to radiolabeled tracer aspreviously described (Riviere et al., 1999).

Pharmacokinetic Analysis of Example 10

Brain concentrations were corrected for residual blood content in theorgan with the equation:C_(Total)=(C′_(Tissue)−(C_(B)*V_(B)))/(1−V_(B)), where C_(Total) is theconcentration of (+)-methamphetamine or (+)-amphetamine in the tissuecorrected for blood concentration; C′_(Tissue) is the tissue drugconcentration before correction for blood content; C_(B) is the drugconcentration in the blood; and V_(B) is the volume fraction of theresidual blood in each tissue (Triplett et al., 1985). V_(B) values forbrain (0.037) were obtained from Khor et al. (1991).

When no mAb was present, the blood drug concentration was assumed to beequal to the serum drug concentration, as (+)-methamphetamine and(+)-amphetamine distribute equally in red blood cells and serum (Riviereet al., 2000). When the mAb was present, it was assumed (due tohigh-affinity mAb binding) that all of the drug was in the serum ratherthan in the red blood cells. The (+)-methamphetamine or (+)-amphetamineconcentration in blood for this calculation was determined bymultiplying each animal's serum drug concentration by 1 minus theirrespective hematocrits (Valentine and Owens, 1996).

To determine the distribution half-lives of (+)-methamphetamine and(+)-amphetamine, the average concentration-vs-time curves were analyzedby model-dependent methods using a nonlinear least-squares fittingroutine. The data were fit to both two- and three-compartment i.v. bolusmodels, with y (predicted concentration), 1/y, or 1/y² weighting. Thebest-fit line was chosen by visual inspection and analysis of theresiduals. The terminal elimination half-life (t_(1/2λZ)) wasdetermined, where possible, from the terminal phase of the averageconcentration-vs-time profiles for (+)-methamphetamine and(+)-amphetamine with the use of model-independent analysis. The areaunder the concentration time curve (AUC) for serum and brain weredetermined from t=38 min (immediately after mAb treatment) to 4.5 h(last measured time point). Because we could not accurately estimate thepharmacokinetic values after mAb (t_(1/2λZ)=8 days, Bazin-Redureau etal., 1997) administration due to the limited period of serum and brainsampling, AUC_(38min) ^(4 5 h) values were used for comparativepurposes. Nevertheless, sufficient data were collected for a comparisonof pharmacodynamic changes during (+)-methamphetamine's pharmacologicaleffect period. All pharmacokinetic analysis was performed with the useof WinNonlin V3.0 (Pharsight Corporation, Mountain View, Calif.).

Statistical Analysis for Example 10

To determine if the administration of the high-affinity mAb affects(+)-methamphetamine-induced locomotor activity, the difference betweenthe baseline (+)-methamphetamine activity (day 7) and(+)-methamphetamine activity after treatment (day 10) was calculated.These differences were then analyzed in a one-way ANOVA context with thedose level of (+)-methamphetamine as the factor. Student's t tests ofthe dose means were carried out, and p-values were adjusted with Holm'scorrection when applicable. These analyses were performed with SASSystem V8.0 software (Cary, N.C.).

For the animals dosed with 1-mg/kg (+)-methamphetamine, comparisonsbetween saline baseline activity (day 1) and a second saline-buffertreatment at the end of the protocol were tested with a paired Student'st-test. To assess mAb-induced changes in (+)-methamphetamine and(+)-amphetamine tissue concentrations at each time point, a Student'stwo-tailed t test was used. These analyses were conducted with SigmaStatV1.0 software (Jandel Scientific, San Rafael, Calif.). A significancelevel of p<0.05 was used for all statistical analyses.

Example 11 Effects of Anti-Meth Fab on Meth-Induced Behavioral Effects

Based on the distance traveled parameter, the duration of action ofmethamphetamine-induced effects following a 1 mg/kg iv dose was abouttwo hours (116±17 min). After treatment with anti-PCP Fab the durationof activity was 111±10 min. After treatment with anti-methamphetamineFab the duration of activity was 75±22 min. Both the distance traveled(FIG. 15A) and the number of rearing events (FIG. 15B) weresignificantly different from the behaviors produced by saline followedby methamphetamine administration (p<0.05). The anti-PCP Fab treatmentproduced some mild reductions in methamphetamine-induced locomotoractivity, which were similar to the mild reductions in behavior we havefound in other experiments in which polyclonal non-specific antibody isused to treat PCP-induced locomotor activity. As a percentage of thecontrol saline treatment, the monoclonal anti-methamphetamine Fabproduced a 55% decrease in the distance traveled (FIG. 15A). The numberof rearing events (FIG. 15B) and the time spent moving (results notshown) were also decreased by 55% and 60%, respectively.

Since the monoclonal antibody used for these studies did notsignificantly bind to d-amphetamine (a psychoactive metabolite presentat very high levels in the rat, but at significantly lower levels in thehuman) and it was a low affinity antibody (about 250 nM), thetherapeutic potential for antibody based medications for overdose arequite significant. This is especially important since no therapiescurrently exist. With the use of improved hapten design and productionof antibodies with significantly lower K_(D) values (e.g., <30 nM), thisinvention should provide a significant breakthrough in treatment ofoverdose due to d-methamphetamine-like drugs.

Example 12 Pretreatment with Anti-(+) Methamphetamine MonoclonalAntibody to Reduce the Effects of (+)Methamphetamine Drug Abuse

Rats (n=7/group) were administered a dose of 502 mg/kg ofanti-methamphetamine monoclonal antibody on day 1. The following daythey were administered i.v. (+)methamphetamine (1.0 mg/kg) 3 days aparton two occasions to stabilize locomotor responses and to minimizesensitization. Then 1.0 mg/kg of (+)methamphetamine was administeredi.v. As shown in FIG. 16, the high-affinity anti-(+) methamphetaminemonoclonal antibody significantly (P<0.05) reduced (+)methamphetamineinduced effects by 42% for distance traveled (left) and by 51% forrearing events (right). The monoclonal antibody significantly shortenedthe duration of action of (+)methamphetamine from about 160 to 80 min.Saline control treatments conducted before and after the experimentalprotocol showed that baseline activity was stable over an extendedperiod.

Example 13 Impact of Anti-D-Methamphetamine Therapy on DrugSelf-Administration and Drug Discrimination as a Measure of TreatingLong-Term Addiction

This example describes the ability of anti-d-methamphetamine antibody toalter the d-methamphetamine dose-response curve for the discriminativestimulus effects of d-methamphetamine, thereby demonstrating possibletherapeutic usefulness of antibody treatment for methamphetamine abuse.

One of the problems in designing experiments to determine if antibodytreatment affects drug discrimination is that antibodies have a verylong duration of action (Proksch et al, 2000). For example, the halflife of a monoclonal IgG in rats is about 8 days (Bazin-Redureau et al,1997). If the same animals are used to determine the effects of drugdoses before and after antibody treatment, the presence of the antibodymight disrupt further drug-discrimination training through antibodybinding of the training dose and reduced access of the training dose tothe brain. Should this occur, maintenance of drug-discrimination controlby (+)-methamphetamine might erode. Therefore, (+)-amphetamine andcocaine were used in addition to (+)-methamphetamine as training drugsin some experiments. These drugs do not cross react with(+)-methamphetamine-specific antibodies and either of these drugs cansubstitute for that drug as a discriminative stimulus. Thus thespecificity of the anti-(+)-methamphetamine antibody should allowcontinued discrimination training using cocaine or (+)-amphetamine asthe training drug.

In addition, the discriminative stimulus effects of (+)-methamphetamine,(+)-amphetamine, and cocaine were compared in rats and pigeons usingseveral routes of drug administration. After determination of thedose-response curves for these drugs, anti (+)-methamphetamineantibodies were given intravenously and all or portions of the(+)-methamphetamine dose-response curves were redetermined. In pigeons,the dose-response curves for (+)-amphetamine and cocaine were determinedbefore and after the administration of antibody to determine the in vivospecificity of an anti (+)-methamphetamine antibody in blocking thediscriminative stimulus effect of (+)-methamphetamine. Dose-responsecurves for (+)-amphetamine were also determined after administration ofthis antibody to rats.

Animal Protocols for Example 13

A total of 16 adult male Sprague Dawley rats were employed. Three ratsperformed poorly during drug discrimination training and two others diedbefore sufficient data were collected. Therefore, the rodent datapresented are based on 11 rats, as shown in Table 2. All rats weremaintained at body weights of approximately 300 g by food pellets earnedduring test sessions, and supplemental feeding after test sessions andon days when the rats were not tested.

A total of 8 male White Carneau pigeons were used in these experiments.Four of the pigeons had performed extensively in previous experiments ondrug discrimination (Li and McMillan, 2001; McMillan et al., 2001b),including experiments in which the discrimination of amphetamines hadbeen studied. These birds were maintained at 80-85% of theirfree-feeding weights (range 429-510 g). The second group of four birdsalso had performed in previous drug-discrimination experiments (McMillanet al, 2001a), although (+)-amphetamine was not used as a training drugin these experiments until the present experiments.

Water was freely available in the home cages of both rats and pigeonsand the vivarium was temperature and humidity controlled with a lightcycle from 0700 to 1900 and a dark cycle from 1900 to 0700. Training andtesting of both species occurred between 0900 and 1200.

Rats were tested in two-lever operant chambers (Gerbrands Model 7400)enclosed in sound-attenuating chambers (Gerbrands Model 7200). Eachchamber contained a house light on the chamber ceiling and stimuluslights over the two levers mounted on the front panel of the chamber. Apellet dispenser delivered 97 mg Noyes food pellets into a cup centeredbetween the levers. Masking noise and air circulation were provided by afan mounted in the rear wall of the sound-attenuating chamber.Programming and recording were accomplished by a MED Associatesinterface and microcomputer located in an adjacent room.

Testing Procedures for Example 13

Rats were conditioned to lever press by autoshaping. After respondingwas established when lights above the right lever were lighted duringone session, responses produced food pellets until 25 pellets had beendelivered. In the next session a similar procedure was followed for theleft lever. Subsequently, the lights above both levers were lighted anddiscrimination training began. Prior to training sessions, rats wereadministered a drug or 0.9% saline solution and placed in the operantchamber for 10 min after which the session began. The different trainingdrugs, doses and routes of administration employed for the three groupsof rats are shown in Table 2.

TABLE 2 Testing Of Rats And Pigeons Before And After mAb6H8Administration GROUP Training Drug and Dose n Testing Condiditons Rat I 2 mg/kg (+) METH^(a) 4 IV and IP (+) METH DRCs pre antibody IV (+) METHdoses days 1 and 4 after antibody IP (+) METH doses on days 1 and 4after second antibody administration Rat II  5 mg/kg Cocaine 3 IV and IP(+) METH 1 day after antibody IV (+) METH days 1 and 7 after a secondantibody administration Rat III 10 mg/kg Cocaine 4 Cumulative IP DRCsfor Cocaine, (+) METH and (+) AMP Cumulative IV DRCs for (+) METH PigeonI  2 mg/kg (+) AMP 4 Cumulative iM DRCs for Cocaine, (+) METH, and (+)AMP IM (+) METH DRCs days 1 and 8 pose antibody Pigeon II  3 mg/kg (+)AMP 4 Cumulative IM DRCs for Cocaine, (+) METH and (+) AMP IM (+) METHDRCs day 2 and 7 post antibody (+) METH = (+)-methamphetamine; (+) AMP =(+)-amphetamine; DRC = dose-response curve; IV = intravenous drugadministration; IP = intraperitoneal drug administration IM =intramuscular drug administration One rat in group Rat I was trainedwith 1 mg/kg rather that 2 mg/kg (+)-methamphetamine

Rats were tested until responding stabilized at more than 90% ofresponding on the drug key after the training drug and less than 10% ofresponding on the drug key after saline. After responding stabilized,dose-response curves were determined for all rats for(+)-methamphetamine by both the intravenous (IV) and intraperitoneal(IP) routes before administration of the antibody. For rats in Groups Iand II, single points on the dose-response curve were determined onTuesdays and Fridays with additional training occurring on other weekdays. For rats in Group III cumulative dose-response curves weredetermined on Fridays, with additional training on other week days.Testing did not occur on Saturday or Sunday.

On test days for Groups I and II, rats were given a dose of drug andthen placed in the test cage for 10 min, after which the session began.The session terminated whenever the animal completed 20 responses on oneof the two levers, or after 40 min, whichever occurred first. On testdays for Group III, rats were treated similarly, except that after thefirst food pellet was consumed, the rats were removed from the testchamber and a second dose was given. The rats were then returned to thetest chamber and 10 min later the session was reinitiated. This processof cumulative dosing continued until the animals no longer responded fora period of 10 min. The cumulative doses shown in the figures representthe sum of all doses given within the session.

The pigeons used in these experiments had performed in experimentspreviously. Those in pigeon Group I had been used most recently in anexperiment where the birds were trained using 4 response keys todiscriminate among saline, 5 mg/kg pentobarbital, 5 mg/kg morphine and 2mg/kg (+)-amphetamine. In these experiments, responses on the correctkey were reinforced under a fixed-ratio 20 schedule of food presentation(Li and McMillan, 2001). These birds continued to be trained and testedin this same chamber, except that training sessions with pentobarbitaland morphine were discontinued, while those with 2 mg/kg(+)-methamphetamine and saline continued.

On training days, (+)-amphetamine or saline was injected into the breastmuscle and the bird was placed in the test chamber for 10 min, afterwhich the chamber and the response keys were illuminated and the sessionwas initiated. Training continued for 40 min or until the pigeons hadreceived 20 reinforcers, whichever occurred first. Once a week, acumulative dose-response curve was determined for cocaine,(+)-amphetamine, or (+)-methamphetamine. In the determination ofcumulative dose-response curves, a dose of drug was administered and thepigeon was placed in the chamber for 10 min, after which the chamber andkey lights were illuminated. The pigeon was tested for 5 min or untilone food reinforcer had been delivered, whichever occurred first. Foodwas delivered following the completion of 20 responses on one of the twokeys. After food had been delivered, or the session had timed out, thebird was removed from the chamber, a higher dose was administered, andthe process was repeated. Cumulative dosing continued until the pigeonfailed to respond during a 5-min period.

In other experiments with some of these pigeons, cumulativedose-response curves were determined for (+)-methamphetamine before, andat 1 and 8 days after, administration of the mAb6H8 antibody (pigeonGroups 1 and 2) or at 1 and 4 days after the administration of themAb6H4 antibody (pigeon Group 2). Because only a few birds were stillavailable for these experiments, the data for days 1-2 and days 7-8 werecombined.

Drugs Used for Example 13

(+)-methamphetamine, (+)-amphetamine and cocaine were purchased fromSigma Chemical Co. as the hydrochloride salts. All doses are expressedas the salts. Drugs were dissolved in physiologic saline solution suchthat doses could be administered in a volume of 0.1 ml/100 g of bodyweight. Injections were given i.v. or i.p. to rats and intramuscularlyto pigeons 10 min before test sessions. The anti (+)-methamphetamineantibody was administered at least one day before determining thediscriminative stimulus effects of drugs.

Data Analysis for Example 13

The percentage of responses on the drug key during training sessionswere averaged across animals and a standard deviation was plotted aroundthese means. Dose-response curves before and after administration of theantibodies were compared using a repeated measures analysis of variance.

Results for Example 13

After responding stabilized, the baseline performance of the threegroups of rats during training sessions was not significantly different,indicating that stimulus control was equivalent across the three groups.Although a two-way repeated measures ANOVA for i.v. dose-response curvesfor (+)-methamphetamine in groups I and II was statisticallysignificant, subsequent Tukeys tests revealed no additional significantdifferences between groups, nor were there statistically significantdifferences in dose-response curves across groups when drugs wereadministered by the i.p. route. Therefore, data from the rats trainedwith (+)-methamphetamine and cocaine were combined for the determinationof dose-response effects to increase group size.

FIG. 17 compares the discriminative stimulus effects of i.p. doses of(+)-methamphetamine, (+)-amphetamine and cocaine in the rats from groupIII. Low doses of all three drugs produced responding on the saline key,while higher doses of these drugs produced responding on the drug key.(+)-Methamphetamine and (+)-amphetamine were nearly equipotent asdiscriminative stimuli, but cocaine was only about one-tenth as potentas the amphetamines.

FIG. 18 shows a comparison between the effects of i.v. and i.p. doses of(+)-methamphetamine conducted in all 11 rats. At low doses of(+)-methamphetamine responding was confined largely to the saline key.As the dose of (+)-methamphetamine increased, responding shifted to thedrug key. Intravenous (+)-methamphetamine was approximately 3 times morepotent than i.p. (+)-methamphetamine.

The top frame of FIG. 19 shows the effects of i.v. (+)-methamphetaminebefore and 1 day after the administration of the low-affinity mAb6H8,and the bottom frame shows data from the same rats before and 4 and 7days after administration of the mAb6H8. Administration of the antibodyshifted the (+)-methamphetamine dose-response curve approximately 3-foldto the right (top frame) on the day after administration of theantibody. A one-way repeated measure ANOVA showed the dose responsecurves to be significantly different. The bottom frame of FIG. 19 showsthe dose-response curves for i.v. (+)-methamphetamine at 4 and 7 daysafter administration of the antibody. Both dose-response curves afterthe antibody were significantly shifted to the right (p<0.05).

FIG. 20 shows the i.p. (+)-methamphetamine dose-response curve beforeand 4 days (top frame) and 7 days (bottom frame) after administration ofmAb6H8. At 4 days after administration of the antibody the(+)-methamphetamine dose-response curve was shifted approximately 3-foldto the right, a shift that was statistically significant (p=<0.05) by arepeated measures ANOVA. By day 7 there was no significant differencebetween the before and after dose response curves.

FIG. 21 shows the effects of intramuscular injections of(+)-methamphetamine, (+)-amphetamine, and cocaine in pigeons trained todiscriminate 2 or 3 mg/kg (+)-amphetamine from saline. Low doses of alldrugs produced responding primarily on the saline key and higher dosesproduced responding on the drug key. (+)-Methamphetamine and(+)-amphetamine were approximately equipotent, while cocaine was aboutone-third as potent as the amphetamines.

FIG. 22 shows the effects of intramuscular doses of methamphetamine inpigeons at 2 and 7 days after administration of mAb6H8. At 2 days afterthe antibody, the (+)-methamphetamine dose-response curve was shiftedslightly downward and to the right relative to its original position. At7 days after mAb6H8 the shift was 3-10 fold.

FIG. 23 shows the effects of intramuscular (+)-amphetamine in pigeons at2 and 7 days after mAb6H8. The dose-response curves at 2 and 7 daysafter mAb6H8 for (+)-amphetamine were not statistically different. Thedose-response curve for responding on the drug key reached a peak at alower dose of (+)-amphetamine 7 days after than it had before theantibody was given.

FIG. 24 shows the effects of i.v. and i.p. cocaine in rats before and 1day after mAb6H8 (top frame) and the effects of i.m. cocaine in pigeonsbefore and 1 day after the antibody (bottom frame). The cocaine curveswere not shifted after administration of the antibody in either rats orpigeons.

FIG. 25 shows the effects of intravenous administration of(+)-methamphetamine to rats before and 1 day after (top frame) and atdays 4 and 7 after the administration of the high-affinity mAb6H4anti-methamphetamine antibody. The antibody shifted the(+)-methamphetamine dose-response curve slightly to the right 1 dayafter its administration. At day 4 the dose-response curve was shiftedto the right by 3-10 fold. By day 10 after administration of the mAb6H4the methamphetamine dose-response curve was close to its originalposition.

FIG. 26 shows the i.m. dose-response curves for (+)-methamphetaminebefore and 1 day after administration of the high-affinity mAb6H4 inpigeons. The antibody shifted the dose response curve to the right byapproximately 10-fold.

These results show that a low affinity (K_(D)=250 nM)anti-(+)-methamphetamine monoclonal antibody (mAb6H8) shifted the(+)-methamphetamine dose-response curve to the right by about one-halflog unit. This effect was robust since it occurred in both rats andpigeons, it occurred after intravenous (rats), intraperitoneal, (rats)and intramuscular (pigeons) routes of (+)-methamphetamineadministration, and it occurred when different training doses of(+)-methamphetamine, cocaine, and (+)-amphetamine were used duringdiscrimination training. A similar shift was shown with the highaffinity mAb6H4 antibody (K_(D)=10 nm).

Although only two rats and two pigeons were available to study thediscriminative stimulus effects of (+)-amphetamine and cocaine beforeand after the administration of the mAb6H8 antibody, the failure of theanti (+)-methamphetamine antibody to shift the dose-response curves forthe discrimination of either of these drugs given by several routes ofadministration suggests that its effects were specific for(+)-methamphetamine. The failure of the antibody to block the effects ofcocaine were observed using two different routes of cocaineadministration in rats (i.v. and i.p.) and by a third route in pigeons(i.m.). Therefore, despite the small number of animals used in theseexperiments, the generality of these findings was established. Although(+)-methamphetamine and (+)-amphetamine differ little in chemicalstructure and in their potency as discriminative stimuli, mAb6H8 has anapproximate 2,000 times lower affinity for (+)-amphetamine, so thefailure of the antibody to shift the (+)-amphetamine dose-response curvewas not unexpected.

Preliminary data suggest that (+)-methamphetamine and (+)-amphetamineare nearly equipotent as discriminative stimuli in the rat. Thisrepresents a potential problem because in rats a considerable fractionof (+)-methamphetamine is metabolized to (+)-amphetamine. In fact, inthe rat peak plasma levels of (+)-amphetamine are obtained within 20 minafter intravenous administration of (+)-methamphetamine (Riviere et al.,1999; 2000). Conversion of (+)-methamphetamine to (+)-amphetamine inrats might explain why the shift in the (+)-methamphetaminedose-response curve was only modest in rats. It is possible that much ofthe (+)-methamphetamine not bound to the antibody was metabolized to(+)-amphetamine. Since the discriminative stimulus properties of(+)-methamphetamine and (+)-amphetamine are difficult to separate, theformation of (+)-amphetamine might have limited the degree to whichmAb6H8 could shift the (+)-methamphetamine dose-response curve in rats.In pigeons, the anti (+)-methamphetamine antibody also shifted the(+)-methamphetamine dose-response curve about one-half log unit to theright. The metabolism of (+)-methamphetamine in pigeons is not known.

The effects of mAb6H8 lasted for a week or longer in both rats andpigeons. The half life of mAb6H8 is 7-8 days. Previous studies withanti-phencyclidine IgG antibodies have shown a functional eliminationhalf-life of 15.4 days, which produced significant reductions in brainphencyclidine for at least 27 days (Proksch et al., 2000). Thebehavioral data suggest an extended functional half-life for theanti-(+)-methamphetamine antibody, but not as long as theanti-phencyclidine MAb.

In these experiments, (+)-methamphetamine, cocaine, and (+)-amphetaminewere used as training drugs, and some times at different doses.Differences in dose-response curves for these drugs that were dependenton the training drug or the training dose were not observed. The factthat the dose-response curve for (+)-methamphetamine in the presence ofmAb6H8 consistently shifted to the right by approximately one-half logunit despite the differences in the training drug and training dose onlystrengthens the generality of the findings.

In the present experiments (+)-methamphetamine and (+)-amphetamine werefound to be equipotent as discriminative stimuli in rats trained todiscriminate 10 mg/kg cocaine from saline. Both the amphetamines wereapproximately 10 times more potent than cocaine (FIG. 17). These dataare similar to previous reports on the relative potency of cocaine andthe amphetamines as discriminative stimuli in rats. In previous studiesin cocaine-trained rats, (+)-methamphetamine and (+)-amphetamine haveranged from 3-30 times more potent than cocaine as a discrimininatvestimulus. In (+)-methamphetamine trained rats, methamphetamine was 10times more potent than cocaine. In pigeons in the present study,(+)-methamphetamine and (+)-amphetamine also were approximatelyequipotent, but the amphetamines were only 2-3 times more potent thancocaine. This is similar to a previous report in pigeons trained todiscriminate cocaine from saline, where little difference was foundbetween the potency of cocaine and (+)-amphetamine. Although the presentstudy found less difference in potency between the amphetamines andcocaine, it is possible that this difference relates to differences inthe training drugs rather than the species in the present study. Therats in which the dose-response curves were determined were trained todiscriminate 10 mg/kg cocaine from saline, while the pigeons in whichthe dose-response curves were determined were trained to discriminate 3mg/kg (+)-amphetamine from saline.

The potency of intravenous and intraperitoneal (+)-methamphetamine wasalso compared in rats. These experiments were performed becausepreliminary experiments with the anti (+)-methamphetamine antibodysuggested that it was more difficult to shift the (+)-methamphetaminedose-response in the presence of the antibody when the(+)-methamphetamine was administered intravenously instead ofintraperitoneally. When more animals were tested, this observation didnot hold up. However, it was established that intravenous(+)-methamphetamine was approximately 3 times more potent as adiscriminative stimulus than intraperitoneal (+)-methamphetamine in ratstrained to discriminate cocaine from saline.

The degree to which the 6H8 anti-(+)-methamphetamine mAb shifted thedrug discrimination curve for (+)-methamphetamine was modest.Presumably, the effectiveness of the antibody would be related to itsaffinity (250 nM), capacity, and the on-off rates of mAb binding to(+)-methamphetamine. The mAb6H8 antibody is a low affinity antibody.Antibodies with significant improvements in the affinity constant andspecificity should be more effective than mAb6H8. The high affinitymAb6H4 did appear to produce a greater shift in the dose-response curvefor (+)-methamphetamine in pigeons than mAb6H8 did. The research withboth mAb6H8 and mAb6H4 illustrates that anti-(+)-methamphetaminemonoclonal antibodies are capable of blocking pharmacological effects of(+)-methamphetamine that are relevant to its abuse.

Example 14 Effect of Antibody-Based Therapy on D-MethamphetamineToxicity in Large Animal Model

A battery of pharmacokinetics studies and behavioral tests can beconducted to determine whether anti-d-methamphetamine Fab can reverseacute behavioral toxicity due to d-methamphetamine in large animals likelarge dogs or primates. These data will help to determine the ability ofanti-d-methamphetamine Fab to redistribute d-methamphetamine in a largeanimal model and help to scale-up the therapy to humans.d-Methamphetamine can be administered to male dogs (or primates; n=6 pergroup, 3 males and 3 females) at 0.3 mg/kg or higher depending onresults of preliminary d-methamphetamine dose-response studies. Ifneeded for quantitation, a tracer dose of [³H]-d-methamphetamine canalso be administered. After the drug is fully distributed (e.g., 30-45min), anti-d-methamphetamine Fab is administered at a 1.0 mol-eq dose tothe amount of d-methamphetamine remaining in the dog (or primate) at 30min. The exact timing and dosing depend on the outcome of the ratstudies and preliminary pharmacokinetic studies in dogs or primates.Plasma and urine d-methamphetamine pharmacokinetics can be determined ineach dog or primate as described above.

The same dogs (or primates) should be used for the pharmacokinetic andbehavioral studies for continuity. However, the success of theexperiments is not dependent on using the same dog (or primate) for allexperiments (n=6). For the behavioral experiments, d-methamphetamine areadministered to dogs (or primates) at 0.3 mg/kg (or higher) followed30-45 min later by a 0.1, 0.3, or 1.0 mol-eq dose ofanti-d-methamphetamine Fab. The experiments are done in a pre-determinedrepeated-measures, mixed-sequence design. The same measures of behavior(and the EthoVision system) as described above can be used in thestudies of d-methamphetamine acute toxicity.

Example 15 Hapten Design and Antibody Selection

When generating monoclonal antibodies (mAb, plural and singular) againstsmall molecules, the chemical composition and molecular orientation ofthe drug-like hapten on the antigen is a crucial determinant, as shownherein. This is especially important when attempting to discovertherapeutic mAb against the drugs of abuse (+)-methamphetamine((+)METH), (+)-amphetamine ((+)AMP) and the related compound(+)-3,4-methylenedioxymethamphetamine ((+)MDMA, the plus isomer in theracemic mixture known as MDMA or ecstasy). The goal of these studies wasto design and synthesize (+)METH-like haptens with structural attributesthat would make them effective for generating monoclonal antibodies fortreating medical problems associated with these stimulant drugs ofabuse.

For these studies, hapten spacers between (+)METH and the carrierprotein were progressively lengthened from 4 to 10 atoms to increase thepotential for greater interaction of the hapten with the antibodybinding site and/or to increase flexibility of the spacer between the(+)METH backbone structure and the carrier. It was hypothesized that aprogressive lengthening and flexibility of the spacer arm would lead toincreased affinity and specificity due to increased access to the entire(+)METH-like structure. As a secondary strategy, the location of thelinker attachment to the (+)METH structure (e.g., para and metaattachments) was varied in an attempt to elicit antibodies withdifferent conformational selectivity for (+)METH-like compounds.

Chemicals and Drugs for Example 15

All chemicals and protein antigens were purchased from Sigma (St. Louis,Mo.), unless otherwise noted. Enzymes and E. coli strains were purchasedfrom Invitrogen (Carlsbad, Calif.). (+)-2′,6′-³H(n)]methamphetamine([³H]-(+)METH; 23.5 Ci/mmol) and (±)-[2,6-3H2(n)]-amphetamine([³H]-(±)AMP; 45 Ci/mmol) were obtained from the National Institute onDrug Abuse (Bethesda, Md.) after synthesis at the Research TriangleInstitute (Research Triangle Park, NC). Other METH-like drugs used inthis study were also obtained from the National Institute on Drug Abuse.

[³H]-(+)METH was used as sent, but the [³H]-(±)AMP waschromatographically separated to obtain [³H]-(+)AMP for use in ourstudies of (+)AMP specificity. The separation was performed on a 150×4mm (i.d.) 5 μm CrownPak CR(+) column (Chiral Technologies Inc., Exton,Pa.). The mobile phase consisted of 0.1 M perchloric acid (FisherScientific) containing 10% (v/v) methanol. The column temperature wasmaintained at 15° C. The flow rate was 1.0 ml/min and the injectionvolume was 50 μL. Chromatographic peaks were detected using ultravioletabsorption detection at a wavelength of 210 nm. The retention times for[³H]-(+)AMP and [³H]-(−)AMP were 20.1 min and 24.4 min, respectively.

Haptens and Hapten-Protein Conjugation for Example 15

Five different stereospecific (+)-isomer (+)METH-like haptens weresynthesized. All haptens were synthesized as HCl salts to aid insolubility, and stored as solids or powders until used. The chemicalstructures are shown in Table 3. The complete synthesis of one of thehaptens ((+)METH P6) was previously reported (Byrnes-Blake et al., 2001,Int Immunopharmacol 1:329-338). Synthesis of an 8 carbon molecule spacerhapten was also attempted, but synthesis of this molecule proved moredifficult than expected, so work on this hapten was postponed till alater date. The chemical names and abbreviations of the five haptensare:

(S)-(+)-4-(3-carboxypropyl)methamphetamine, (+)METH P4

(S)-(+)-4-(5-carboxypentyl)methamphetamine, (+)METH P6

(S)-(+)-4-(5-carboxypentyloxy)methamphetamine, (+)METH P06

(S)-(+)-3-(5-carboxypentyloxy)methamphetamine, (+)METH M06

(S)-(+)-3-(9-carboxynonyloxy)methamphetamine, (+)METH M010

Each hapten was initially covalently bound to at least 2-3 differentprotein antigens and used for immunization of mice to test for anti-METHIgG response. The individual mouse and hapten-protein antigencombination that yielded the highest anti-(+)METH IgG titers was chosenfor production of monoclonal antibodies (see details below). Thefollowing is a list of the hapten-protein conjugates that produced themAb listed in Table 3: (+)METH P4 and (+)METH P6 conjugated to bovineserum albumin; (+)METH PO6 and (+)METH MO6 conjugated to ImjectSupercarrier Immune Modulator (catonized BSA (cBSA), Pierce Biotech,Rockford, Ill.); (+)METH MO10 conjugated to ovalbumin (OVA).

All chemical reactions for covalent binding of the haptens to proteinantigens followed the same general procedure. The haptens were firstsolubilized in either 0.1 M 2-[N-morpholino]ethanesulfonic acid buffer(pH 4.5) or dimethylformamide and then adjusted to pH 4.5 with HCl. Allhaptens were coupled to their respective protein antigens by acarbodiimide reaction using the cross-linker1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (PierceBiotech). This chemical synthesis forms a peptide bond between thecarboxyl group of the hapten linker arm and free amino groups of lysineside chains in the respective proteins. The reactions were conductedwith continuous stirring under dark conditions at room temperature for18 hrs. At the end of the reaction, all antigens were purified asdescribed by Byrnes-Blake et al. (2003, EurJ Pharmacol 461:119-128).This purification involved dialysis against distilled water,phosphate-buffered saline (pH 7.4), and a final purification of thesoluble fraction on a gel filtration column in phosphate-buffered saline(pH 7.4). Purified antigens were stored at −20° C. until needed.

Immunization, Screening, and Hybridoma Generation for Example 15

Female BALB/c mice (Charles River Laboratories, Wilmington, Mass.) wereused for all immunizations. For production of the (+)METH P6 mAb, micewere immunized subcutaneously in the hindquarters with 100 μg of the(+)METH P6 antigen emulsified 1:1 (v/v) in TiterMax adjuvant (CytRxCorporation, Norcross, Ga.) and boosted monthly with 50 μg of theantigen until a favorable titer was reached. For all other antigenimmunizations, the mice were initially immunized in the hindquarterssubcutaneously with 20-100 μg of antigen emulsified in Freund's completeadjuvant. The initial immunization was followed by a boost with 20-50 μgof antigen emulsified in Freund's incomplete adjuvant three weeks laterfollowed by three boosts at six week intervals, until a favorable titerlevel was reached. Serum samples were taken via tail bleed periodicallyto measure anti-(+)METH IgG. Titers were measured by ELISA(enzyme-linked immunosorbant assay) using 96-well microtiter platescoated with the original hapten conjugated to a different protein. Forexample, if the original antigen was (+)METH-MO6-cBSA, (+)METH-MO6conjugated to thyroglobulin was used to avoid selecting carrierprotein-reactive antibodies. The screening for anti-(+)METH IgG responsewas conducted by a [³H]-(+)METH radioimmunoassay (RIA), using (+)METHand (+)AMP as the inhibitors. After sufficient anti-(+)METH IgG titerswere achieved, conventional hybridoma technology was utilized asdescribed previously (Valentine et al., 1994, J Pharmacol Exp Ther269:1079-1085). The hybridoma fusion partner for mouse B cells was cellline P3X63Ag8.653 (American Type Culture Collection, Manassas, Va.). IgGisotype and light chain identity was determined with a mouse antibodyisotyping kit (Boehringer Mannheim, Indianapolis, Ind.).

Production and Purification for Example 15

Monoclonal antibodies were produced in either a Cell-Pharm System 2500hollow fiber bioreactor (Valentine et al., 1996, J Pharmacol Exp Ther278:709-716); Unisyn Technologies, Inc., Hopkinton, Mass.) or in aBiostat B 10 liter bioreactor (Sartorius Corp, Edgewood, N.Y.). Allantibodies were harvested and stored at −80° C. until purification. MAbwere purified either by affinity chromatography using Protein-GSepharose (Amersham Biosciences, Piscataway, N.J.), or ion exchangechromatography using SP Sepharose (Amersham Biosciences, Piscataway,N.J.) as described in Hardin et al. (1998, J Pharmacol Exp Ther285:1113-1122), ora combination of the two methods. Followingpurification, all antibodies were concentrated and buffer exchanged into15 mM sodium phosphate containing 150 mM sodium chloride (pH 6.5-7.5) asdescribed in McMillan et al. (2002, Behav Pharmacol 13:465-473).

Determination of Immunochemical Specificity.

The cross-reactivity profiles of each mAb for methamphetamine,structurally related, and unrelated compounds, was determined by RIA ina manner similar to that described by Owens et al. (1988, J PharmacolExp Ther 246:472-478). An IC50 value for inhibition of [³H]-(+)METH (and[³H]-(+)AMP for the mAb generated against the (+)METH MO10 hapten) wasdetermined for each ligand after fitting a sigmoidal curve to the datapoints. K_(D) values for mAb were determined by the method of Akera andCheng (1977, Biochim Biophys Acta 470:412-423).

Results for Example 15

For these studies, the hapten spacers were progressively lengthened from4-10 atoms to increase the potential for greater interaction of theMETH-like structures with the antibody binding site and to increase theflexibility of the spacer. It was hypothesized that a progressivelengthening of the spacer arm would lead to increases in affinity due toimproved access to the entire METH-like structure; and the differentimmobilized conformations would elicit antibodies having differentconformational selectivity for (+)METH-like compounds.

The haptens were conjugated to the terminal amino groups of lysines inbovine serum albumin or ovalbumin by carbodiimide chemistry, which formsa peptide bond with the available carboxylic acid on the hapten. Therewere 59 lysines in bovine serum albumin, 20 in each of the four subunitsof ovalbumin and even more conjugation sites were available oncationized bovine serum albumin (i.e., Imject Supercarrier ImmuneModulator). However not all of the lysines or conjugation sites wereavailable at the surface of the protein for coupling to the haptens.Preliminary optimization experiments showed that a ratio of hapten toprotein of 30:1 to 90:1 yielded the best incorporation rates for thesyntheses. While the hapten incorporation rate for the antigens couldnot be precisely determined, initial mass spectrophotometry studiesindicated that an average of 4 haptens were conjugated to each moleculeof protein.

Because the primary goal was to select for high-affinity mAb, theantigen dose was kept relatively low (e.g., 10-20 μg). Whileimmunization with higher hapten-antigen doses (e.g., 50-100 μg)sometimes led to higher titers, the affinity for (+)METH was often toolow. Thus a minimum dose of antigen was typically used. This strategyroutinely led to immunological response in only 40-70% of animals. Inmore recent studies, it was discovered that a primary reason for <100%immunological response was the low incorporation rate of the hapten onprotein antigens, which in part was overcome by judicious use ofFreund's complete and incomplete adjuvants to boost and sustainimmunological response.

Each mouse serum from each group of immunizations was routinely screened(typically 6-10 mice) after each boost to determine the maturity of theimmune response and the relative immunochemical characteristics of thepolyclonal serum (titer, affinity and specificity). For this, a[³H]-(+)METH RIA was used. The screening assay always involvedinhibitions of [³H](+)METH binding with increasing doses of (+)METH and(+)AMP to determine the relative affinities for each ligand. The finalchoice of a specific mouse for use in generating hybridomas was basedprimarily on the animal with the highest titer and affinity for (+)METH.From this process of screening immune serum, 3-10 unique monoclonalantibodies were generally found from each fusion. Most importantly, apolyclonal antiserum that was positive for (+)AMP was not discovereduntil the MO10 hapten was used.

For producing the hybridomas, mice were chosen that had been immunizedwith Freund's complete adjuvant and boosted with Freund's incompleteadjuvant. The one exception was the immunizations with (+)METH P6, whichused Titermax as the adjuvant. In preliminary optimization experiments,immunizations with alum precipitated antigens, Titermax adjuvant andRibi's adjuvant were tried on several occasions. While these adjuvantsgenerally produced high titers, it was found that the highest affinityantibodies were generated with Freund's adjuvants.

Example 16 MAb Cross Reactivity Studies

After screening over 25,000 potential hybridoma cell lines for mAbproduction, five mAb with the most favorable immunochemicalcharacteristics were extensively studied for molecular properties andpreclinical efficacy (see Table 3). The rest of the hybridoma cell lineswere stored frozen in case of future need. The selection of a mAb formore extensive in vitro and in vivo testing was based on the desire tohave a range of affinities, a range of drug specificities, and a highlevel of mAb production from the parent hybridoma cell line. This finalcriterion was needed to increase the feasibility of large scale mAbproduction for in vivo testing. In most cases there was one or moresimilar affinity or specificity mAb that were produced from the samefusion. For instance, the separate fusions that produced mAb6H4 andmAb4G9 (see Table 3) also produced mAb with virtually the same affinityand specificity, but slightly different amino acid sequences. These twoparticular antibodies were chosen because the parent hybridoma cell lineproduced significantly more mAb.

TABLE 3 Chemical Structure of Haptens, the Resulting mAb, and K_(D)Values for Key Drugs mAb Name Key Psychostimulants (Isotype and (+)METH(+)AMP (+)MDMA Hapten Structure Hapten Name light chain) K_(D) (nM)K_(D) (nM) K_(D) (nM)

(+)METH P4 mAb6H8 (IgG₁ κ) 250 41,000 106

(+)METH P6 mAb6H4 (IgG₁ κ) 11   4000 4

(+)METH PO6 mAb6H7 (IgG_(2b) κ) 95 47,000 87

(+)METH MO6 mAb9B11 (IgG₁ λ) 41   5000 123

(+)METH MO10 mAb4G9 (IgG_(2b) κ) 34   120 (51 nM with [³H]-(+)AMP) 140

Results for Example 16

RIA was used to determine the relative affinity and cross-reactivityprofile of each mAb (Tables 3 and 4). Only one of five haptens generatedmAbs with the desired therapeutic potential. Immunization with the MO10hapten resulted in production of mAb (mAb4G9) with high-affinity bindingto (+)METH, (+)AMP, and (+)MDMA; little or no cross-reactivity with(−)METH-like isomers; and no significant cross-reactivity withendogenous compounds or structurally similar common medications (Tables3 and 4). No other hapten/linker location yielded an antibody with highaffinity for all three drugs of abuse.

TABLE 4 Characterization of the binding specificities of three importantprototype anti-METH/MDMA or anti-METH/MDMA/AMP mAb. Antibody Specificity(Relative Potency to METH)^(a) mAb6H4 mAb6H8 mAb4G9 Drug (11 nM)^(b)(250 nM)^(b) (34 nM)^(b) (+)METH 1.00 1.00 1.00 (+)AMP 0.001 0.023 0.34(+)MDMA 1.25 3.40 0.29 (−)METH 0.030 0.011 0.102 (−)AMP <0.001 0.0030.063 (−)MDMA 0.007 0.018 0.011 (+)MDA 0.001 0.024 0.090 (−)MDA <0.001<0.001 0.002 4-OH-METH 0.588 0.294 0.106 (+)pseudoephedrine <0.001 0.0180.004 (+)norpseudoephedrine <0.001 <0.001 <0.001 l-phenylphrine 0.001<0.001 <0.001 (+)ephedrine <0.001 <0.001 <0.001 (+)phenylpropanolamine<0.001 <0.001 <0.001 β-phenylethylamine <0.001 <0.001 0.001 tyramine<0.001 <0.001 <0.001 dopamine <0.001 <0.001 <0.001 norepinepherine<0.001 <0.001 <0.001 serotonin <0.001 <0.001 <0.001 epinephrine <0.001<0.001 <0.001 ^(a)Relative potency to METH = (RIA IC50 value forMETH/RIA IC50 value for test ligand). See Table 3 for the structures ofthe haptens used to generate these antibodies. ^(b)IC50 value for METHbinding from Table 3.

Since mAb4G9 was the only mAb to significantly cross-react with (+)AMP(Tables 3 and 4), its affinity for (+)AMP was examined in more detail.For this, a RIA analysis was conducted using [³H]-(+)AMP (in addition toa RIA with [³H]-(+)METH) and AMP as the inhibitor. These data showed theactual affinity for AMP was 51 nM (Table 3), demonstrating that this mAbhas virtually the same K_(D) value for AMP and METH. [³H]-(+)MDMA wasnot available for determining a more accurate K_(D) value for (+)MDMAbinding, but it seems likely that the true K_(D) value would besignificantly lower than the value indicated by MDMA inhibition of[³H]-(+)METH binding in the RIA.

Attaching the linker of the hapten distal to the chiral center of themolecule yielded a refined specificity for (+)-isomers (Table 4). Therelatively short length of spacer arms of haptens (+)METH P4 and (+)METHP6 (4- and 6-carbon linkers, respectively), coupled with attachments atthe para-carbon of the (+)METH phenyl ring (Table 3), hindered theflexibility of haptens. This likely forced the immune system torecognize the presence of the methyl group on the nitrogen molecule of(+)METH and (+)MDMA and its absence in (+)AMP. Thus, mAb affinity washigh for (+)METH and (+)MDMA, but low for (+)AMP. The hapten (+)METHPO6, like (+)METH P6, was designed with a linker attached to thepara-carbon of the phenyl ring, but an oxygen was included to influencelocalized charge and solubility and mimic the presence of one of twooxygen atoms at the para and meta positions of the methylenedioxy groupof (+)MDMA (Table 3). An oxygen attached to the phenyl ring structurewas included in two other haptens, (+)METH MO6 and (+)METH MO10, butlinkers were attached to the meta-carbon of the phenyl ring of (+)METH.This strategy was designed to present the oxygen of the (+)MDMA-likestructure along the same spatial plane as the (+)METH molecule's chiralcenter. The longer (+)METH MO10 spacer was used to allow moreflexibility in the hapten on the protein in hopes of discovering mAb(s)with broader recognition of (+)METH-like structures. These combinedstrategies resulted in the best balance of affinity and specificity.

From these studies, it was learned that 1) linkers located distal to thechiral center of this very small molecule favor generation ofstereospecific antibodies, 2) a longer flexible linker arm like (+)METHMO10 favors generation of antibodies with broader selectivity for(+)METH-like compounds, and 3) spacers ≧6 atoms produce higher affinitymAbs. Importantly, discovery of mAb4G9 was not an isolated event, asother MO10-derived mAbs with similar specificities for (+)METH and(+)AMP have since been discovered.

Example 17 Antibody Sequence Analysis

To gain a better molecular understanding of how the primary amino acidsequence affected mAb affinity for (+)METH, related and unrelatedsequence features in each mAb variable region was analyzed. Three of themAb were IgG1 subclass and two were IgG2 (Table 3). Except foranti-METH/MDMA mAb9B11 (λ light chain), all of the mAb possessed a κlight chain.

cDNA Cloning and Sequencing of mAb for Example 17

For these studies, five prototype anti-METH mAb ranging in METHaffinities from 11 to 250 nM were analyzed (Table 3). A single prototypemAb resulting from each of the haptens was chosen for detailed studies.The light chain (LC, singular and plural) cDNA of the mAb were cloned byRT-PCR using Superscript II reverse transcriptase (Invitrogen) with anexact reverse primer matching C-terminus of the light chain namedMLEND1.Not (5′-GGG GCG GCC GCG CGT CTC AGG ACC TTT GTC TCT AAC-3′) (SEQID NO:1). The light chains of mAb6H4, mAb6H8, and mAb6H7 were amplifiedin the forward direction with the degenerate primer ML2, and the lightchain of mAb4G9 was amplified in the 5′ direction with the degenerateprimer ML4 (Coloma et al., 1992). The light chain of mAb9B11 wasamplified in the forward direction with the primer sequence5′-ATGGCCTGGA(T/C)TTCACTTATACTCTCTCTCCTGGCTCTC-3′ (SEQ ID NO:2). Theresulting cDNA was blunt-ligated into the Sma I site of the cloningvector pGEM-3Z.

The heavy chain cDNA of all IgG1 (from (+)METH P4, (+)METH P6 and(+)METH MO6) mAb were amplified using RT-PCR as described above with anexact reverse primer to the C-terminus of the heavy chain, namedMHEND.NotI 5′ GGG GCG GCC GCA GGG CTC CM GGA CAC TGG GAT CAT TT 3′ (SEQID NO:3), and a mixture of three degenerate primers based on the MHALTprimers from Coloma et al. (1992, J Immunol Methods 152:89-104). Theprimers were modified from the originally published sequence only by thesubstitution of a Nhe I restriction site for the original restrictionsite. The IgG2 mAb (from (+)METH PO6 and (+)METH MO10) were amplifiedwith the reverse primer 5-CTCCCGGTCTCCGGGTAAATGA-3′ (SEQ ID NO:4).

The forward sequence of the heavy chain of mAb6H8 was amplified withprimer MHALT1 (Coloma et al., 1992, J Immunol Methods 152:89-104). Theforward primers for mAb6H4, mAb6H7, mAb9B11, and mAb4G9 were designedfrom the results of N-terminal sequencing of the mature proteins (seeFIGS. 27A and B for protein sequences). The primer sequences used were:5′-GAGTGCAGCTTCAGGAGTCAGGACCTAGC-3′ (SEQ ID NO:5) formAb6H4,5′-GATGTAAAACTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTC-3′ (SEQ IDNO:6) for mAb6H7,5′-GAGGTGCAGCTTCCGGAGTCAGGACCTAGC-3′ (SEQ ID NO:7) formAb9B11, and 5′-GAGTACCAGCTCCAGCAGTCTGGGAC-3′ (SEQ ID NO:8) for mAb4G9.The cDNA was then blunt-ligated into the Sma I site of cloning vectorpGEM-3Z. The resulting plasmids of all mAb cloning was transformed intoE. coli strain DH5a and sequenced at University of Arkansas for MedicalSciences DNA Core Sequencing Facility.

All sequences were submitted to the GenBank database. TheGenBank-assigned accession numbers of the light chains of mAb6H8,mAb6H4, mAb6H7, mAb9B11, and mAb4G9 are 774083, 786626, 877567, 881246,and 877579 respectively. The GenBank-assigned accession numbers of theheavy chains of mAb6H8, mAb6H4, mAb6H7, mAb9B11, and mAb4G9 are 774081,774071, 881226, 877571, and 877573, respectively. The germ-line usage ofthe different mAb was determined by comparing the DNA sequences to thosein the IMGT database using the web-based program V-QUEST tools (Internetaddress: http://imgt.cines.fr) and by visual examination of thesequences (Giudicelli et al., 2004, Nuc Acid Res 32:W435-440).

Results for Example 17

Alignments of the amino acid sequences of the variable region of the mAbis presented in FIG. 27. An analysis of complementary determiningregions revealed a high degree of diversity in both composition andlength. The first light chain CDRs (L1) varied in length from 10-14residues, and with the exception of mAb4G9, possessed a large number ofserine residues (FIG. 27B). The only conserved residue in CDR L1, or anyof the light chain CDRs, was the serine at position L26. The L2 CDRswere 7 residues in length except for mAb6H7, which possessed only 5amino acids. The L3 CDRs were all 9 residues in length except mAb4G9which had 10 residues. The CDRs of the heavy chain regions (FIG. 27A)exhibited similar lengths in CDRs H1 and H2, but little homology. CDR H1had a conserved threonine at position H30 and either a tryptophan ortyrosine at position H33. CDRH3 differed in length from 8-16 residues.Although not immediately apparent from the alignment, all H3 regionspossessed two tyrosine residues spaced five residues apart, with thesecond tyrosine before the tryptophan at H103.

While comparisons of CDR sequences are important, differences in CDR canbe attributed to differences in germ-line sequences of particularV-region genes, and to somatic mutation within the CDRs of theseV-region genes. To better understand the relative importance of thegerm-line and somatic mutations, the sequenced genes were analyzed usingthe IMGT database (Giudicelli et al., 2004, Nuc Acid Res 32:W435-440).The analysis showed that each antibody was unique and not clonal. Thatis, rather than coming from one germ-line gene arrangement early in Bcell development, they resulted from unique V(D)J recombination events.These unique germ-line gene rearrangements then underwent somatic DNAmutations, that were often silent, but some resulted in amino acidchanges that differed from the original germ-line gene. Thus, no clearpattern of response was found.

This sequence analysis elucidated unique sequence differences in theantibody CDRs. A common feature was a conserved proline at position 95or 95a of all CDR L3 regions, except for mAb9B11 (FIG. 27B), which hadserine residue. Because of their ability to form “hinges,” prolineresidues often lend flexibility in main chain protein sequences. Thisproline/serine was immediately followed by either a hydrophobic aminoacid (i.e. leucine or valine as in mAb6H4 and mAb9B11, respectively) oran aromatic residue. It is possible that these residues could beimportant for interaction with the phenyl ring of (+)METH-like compoundsvia hydrophobic or pi-pi interactions, and the preceding proline couldlend flexibility to adapt to different conformations.

Example 18 Molecular Modeling and Docking

Based on the results of the primary sequence alignment, three mAb(mAb6H4, mAb6H8 and mAb4G9) were chosen for structural modeling. EachCDR variable region was assigned and given a canonical classification(Al-Lazikani et al., 1997, J Mol Biol 273:927-948), except for the H₃CDRs, which do not possess canonical classes.

IgG Variable Region Structural Modeling and Analysis for Example 18

Molecular modeling of the three dimensional structure of the variableregions of three of the mAb was performed using the WAM antibodymodeling algorithm (Whitelegg and Rees, 2000, Protein Eng 13:819-824).mAb6H4, mAb6H8, and mAb4G9 were chosen for more detailed analysisbecause they exhibited the full range of affinities for (+)METH and abroad range of ligand specificities for other important METH-like drugs.The primary amino acid sequences of the variable regions of the HC andLC were first submitted to the WAM antibody modeling site for alignment.The program aligned the sequences against known sequences in thedatabase and searched for canonical classes of complementary determiningregions (CDR). Based on these classifications, the program assigned a3-dimensional structure to the framework and CDR regions by fitting themain chain to that of the closest known structures.

Ligand Docking for Example 18

For docking simulation, the FlexX (Tripos) program was used. First, adeep pocket was identified at the interface of the CDR regions fromsurface modeling and electrostatic calculations in Pymol (DelanoScientific, San Carlos, Calif.) and Sybyl (Tripos). To define thisregion as a putative active site, residues within an area 6 Å around FL94 (for mAb6H4) or Y L94 (for mAb4G9) were selected. The METH ligandwas assigned formal charges by Sybyl and the molecule was allowedpartial flexibility. The program was set to find the 30 best dockingconformations and return these in a consensus scoring table.

Results for Example 18

The three-dimensional models exhibited classical antibody β-sheet foldconformation (FIG. 28). In general, all models showed conformity withgeometrical constraints throughout the structures. The analysisindicated that less than 2% of residues had main chain Ψ and Φ angles inoutlier regions. All three models appeared to conform reasonably well toknown protein structural features and constraints, and they presented anappropriate foundation to conduct base docking analysis.

All CDRs fell within canonical classes except L3 of mAb4G9 and theH3CDRs, which do not have canonical classes. The CDR H3 regions of allthree antibodies were predicted to form a kinked or “hairpin,” ratherthan extended conformation. Comparison of the models revealed conservedstructural elements and some potentially important differences in theroot mean square deviations (RMSD) of the CDR loop configurations (FIG.29). The loop structure of mAb6H4 was arbitrarily chosen as a referencepoint to compare the differences from the other two antibodies, becauseit had the highest affinity for (+)METH. The L2 CDRs of all threeantibodies occupied nearly the same spatial positions. The L3 regions ofmAb6H4 and mAb6H8 were very similar, even though they differed inaffinity for (+)METH by about 25-fold.

Based on the modeling results, docking simulation was performed withmAb6H4 and mAb4G9. According to the models (FIG. 29), a deep pocket wasformed by the interaction of CDR loops H1, H2, L1 and L3 for bothantibodies, with a wider pocket formed in the binding region of mAb4G9due to a shorter H3CDR. A theoretical docking of (+)METH was createdinto these mAb pockets and identified residues within 8 angstroms of theligand as possible sites for ligand-mAb interaction (FIG. 29). Theresults of this FlexX-based docking indicated that the METH molecule wasgenerally oriented with the hydrophobic phenyl group toward the interiorof the pocket. In mAb6H4 and mAb4G9, the charged nitrogen of METH was inclose proximity to a histidine at position L32 and H35 respectively.

Based on the molecular modeling analyses, the interface between (+)METHand the mAb was relatively small, (the surface area of (+)METH is 174 Å2) with small shifts in protein conformation producing large changes inbinding. As can be seen in FIG. 28, the most striking deviationsappeared in the H3CDR region, with over 6 Å and 7 Å RMSD in mAb6H8 andmAb4G9, respectively. The diversity in the positions of the CDR suggeststhat each of these antibodies exhibited a binding paradigm to(+)METH-like drugs that was somewhat independent of loop configuration.The surface rendering of the models exposed a deep pocket at the CDRinterface of mAb6H4. This pocket appeared to be approximately the sizeof (+)METH and would likely accommodate docking of the ligand. Bycontrast, the potential binding pocket of mAb4G9 was wider andshallower. It is hypothesized that the longer linker arm of (+)METH MO10combined with the changed dihedral angle of an oxygen at the metaposition of the phenyl ring contributed to the formation of a largerpocket. Analysis indicated that only five of the six CDR loops, might bedirectly involved in binding of (+)METH-like drugs, with L2 showinglittle contact. The (+)METH docking simulation with FlexX indicated thatthe potential binding pockets were dominated by aromatic residues withsome capable of making hydrogen bonds (i.e., histidine and tyrosine).

Example 19 Concurrent (+)Meth Use and Immune Response

Immunization with (+)METH P6-KLH conjugate was examined in more detailto determine if active immunization would result in anti-(+)METHantibodies and if chronic (+)METH use would affect production ofanti-(+)METH antibodies. These questions are important becausegeneration of an antibody response is dependent on specific immunereceptor recognition of (+)METH-conjugates, and patient use of (+)METHduring immunization could block an immune response.

For these studies, male Sprague-Dawley rats were immunized with KLH(control group) or the (+)METH P6 hapten-KLH conjugate (see Table 3 forhapten structure). (+)METH P6-KLH animals were further divided into twoimmunized groups—one with no subsequent administration of (+)METH, theother repeatedly challenged with (+)METH (3 mg/kg ip; twice a week).Analysis of relative antibody affinities was accomplished in an ELISA byadding increasing concentrations of (+)METH to mouse immune serum inmicrotiter plate wells coated with a (+)METH P6-ovalbumin conjugate.

By this measure, both groups of (+)METH P6-KLH immunized rats developedand maintained anti-(+)METH antibody titers throughout the 53-dayimmunization period (FIG. 30, open and closed circles) compared withcontrol KLH-immunized rats, which had no response (squares). Repeatedadministration of (+)METH to immunized animals did not affectdevelopment or maintenance of anti-(+)METH titers (open circles),compared to immunized rats without a (+)METH challenge. In determiningif there was a relative change in the serum antibody affinity for(+)METH in rats receiving repeated (+)METH administration, there was nodifference in relative antibody affinity for (+)METH betweennon-challenged and (+)METH-challenged groups (FIG. 31).

Thus, challenging rats by repeated administration of (+)METH during thestudy did not affect antibody affinity constants for (+)METH or antibodyserum titers. These studies demonstrate that chronic (+)METH use doesnot interfere with the quantity (titer) or quality (specificity,affinity) of the anti-(+)METH antibody response. These are importantfindings because many addicted patients will likely use (+)METH duringtheir active immunization treatments.

Example 20 Anti-(+)METH mAb Alter (+)METH Pharmacokinetics in Rats

The ability of anti-(+)METH mAb6H4 (generated against (+)METH P6 hapten,see Table 3) to alter (+)METH brain concentrations was examined in twodifferent models of (+)METH abuse (Byrnes-Blake et al., 2005, Eur JPharmacol 521:86-94).

The overdose model was designed to mimic a drug abuser taking a high iv(+)METH dose and treated with (+)METH mAb in the emergency room. In thismodel, rats received 1 mg/kg (+)METH (iv) followed 30 min later by ananti-(+)METH mAb dose. The mAb pretreatment model was designed to mimican abuser in drug treatment administered an anti-(+)METH mAb medicationat the start of behavioral modification therapy who relapses to (+)METHuse. In this model, rats were pretreated with anti-(+)METH mAb6H4 andreceived a 1 mg/kg iv (+)METH dose the following day. This dose (withoutmAb) produced about 2.5 hrs of locomotor effects. Rats (3/time point)were sacrificed at varied times after (+)METH administration todetermine (+)METH brain concentrations. As shown in FIG. 32, mAb6H4decreased (+)METH brain concentrations in both models. Indeed, (+)METHbrain concentrations in both models were virtually superimposable atcomparable times after 30 min—the time of mAb administration in theoverdose model. Both studies clearly show that antibodies against(+)METH can significantly reduce (+)METH brain concentrations over time.

Next, the “functional” half-life of each of the afore-mentionedanti-(+)METH mAbs (see Table 3) was determined. This “functional” assaycompared (+)METH concentrations in the absence and presence of mAbs. Bythis measure, the best antibodies are those with the highest and longestincreases in serum (+)METH and (+)AMP concentrations. First, it wasdetermined that the pharmacokinetic properties of the mAbs were notdifferent (results not shown). For instance, they all had a serumhalf-life of about 7-8 days, which ruled out the possibility that one ormore of them were quickly cleared and thus inactivated throughelimination. It also showed the potential to produce a long-actinganti-(+)METH therapy by passive or active immunization.

To conduct the “functional” studies, male rats (n=4/group) were given14-day continuous (+)METH infusions at 5.6 mg/kg/day by sc osmoticminipumps. After achieving steady state (+)METH concentrations (at 24hrs), each rat was treated with a dose of mAb that was equimolar inbinding sites to the steady-state body burden of (+)METH. Only a singledose of mAb was administered at this time point, but (+)METH wascontinuously infused at a rate of 50% of the body burden per hour tomaintain a (+)METH steady state. Serum samples were collected pre-mAband at time points after mAb administration. All anti-(+)METH mAb causedsignificant acute increases in serum (+)METH concentrations comparedwith pre-mAb controls. However, there were substantial differences inserum (+)METH concentration vs. time curves for the five mAbs (FIG. 33,open symbols). Most anti-(+)METH mAbs appeared to be partiallyinactivated to differing degrees over time, as judged by their inabilityto maintain high concentrations of (+)METH in serum over time. Thisinactivation was particularly striking for the highest affinity mAb6H4(K_(D)=11 nM). However, mAb4G9 ((+)METH and (+)AMP, K_(D)=34 and 51 nM,respectively) was still very effective after about 2 wks. It was alsothe only mAb that maintained significantly increased concentrations of(+)AMP (closed circles) and (+)METH (open circles) over time compared topre-mAb concentrations (square symbols with “A” and “M” inside).

Example 21 Clearance in Brain and Serum

It was originally hypothesized that mAb affinity was the primary drivingforce for therapeutic efficacy. However, these studies revealed that theduration of action and function of the anti-(+)METH mAb in vivo wasdecreased based on predictions from known mAb pharmacokinetics, whichwas unanticipated. The first generation of haptens (e.g., (+)METH P6 and(+)METH P4) were purposely designed to produce mAbs specific for(+)METH, with virtually no cross reactivity with (+)AMP. When the secondgeneration of haptens (e.g., (+)METH MO10) were produced withspecificity for (+)METH and (+)AMP, it was discovered that the resultingmAb (mAb4G9) had the other advantages of increased duration of actionand efficacy.

For instance, see FIG. 37. mAb4G9, unlike mAb6H4 (generated by the P6hapten), alters the distribution of AMP. As shown, mAb4G9 reduces bothMETH and AMP concentrations in the brain, which is medically important.It also repartitions AMP into the serum from other compartments (FIG.36). mAb6H4 has little to no effect on AMP in either brain or serumbecause it is not broadly specific.

Furthermore, mAb4G9 has a longer functional half-life than mAb generatedby previous haptens. FIG. 36 shows mAb6H4 has significantly less in vivofunctionality (increased serum levels of METH/AMP is an indication ofactivity) by the 24 h timepoint. In contrast, mAb4G9 maintainssignificantly elevated serum METH and AMP concentrations 24 h afteradministration, indicating extended in vivo functionality.

The following, while not intending to be exhaustive, lists referencescited herein:

-   -   Bazin-Redureau et al., J Pharm Pharmacol 49:277-281 (1997);    -   Byrnes-Blake et al., Int Immunopharmacol 1:329-338 (2001);    -   Cho et al., Synapse 39:161-166 (2001);    -   Collings, Cable News Network Feb. 13 (1996);    -   Cook et al., Drug Metab Dispos 21:717-723 (1993);    -   Davis and Preston, Anal Biochem 116:402-407 (1981);    -   Goding, Monoclonal Antibodies: Principles and Practice, p.        118-122, Academic Press, New York (1983);    -   Hardin et al., J Pharmacol Exp Ther 285:1113-1122 (1998);    -   Khor et al., Drug Metab Dispos 19:486-490 (1991);    -   Laurenzana et al., Drug Metab. Dispos. 23:271-278 (1995);    -   Li and McMillan, Behav Pharmacol 12:621-628 (2001);    -   McMillan et al., Behav Pharmacol 12:195-208 (2001a);    -   McMillan et al., Pharmacol Biochem Behav 68:395-402 (2001b);    -   Minh-Tam et al., Anal. Biochem. 116:402-407 (1981);    -   Owens et al., J. Pharmacol. Exp. Ther. 246:472-478 (1988);    -   Proksch et al., J Pharmacol Exp Ther 292:831-837 (2000);    -   Rowland and Tozer, Clinical Pharmacokinetics: Concepts and        Applications, 3rd ed, Williams & Wilkins, Baltimore (1995);    -   Riviere et al., J Pharmacol Exp Ther 291:1220-1226 (1999);    -   Riviere et al., J Pharmacol Exp Ther 292:1042-1047 (2000);    -   Tempest et al., Biotechnology 9:266-271, (1991);    -   Triplett et al., J Pharm Sci 74:1007-1009 (1985);    -   Valentine et al., J Pharmacol Exp Ther 278:709-716 (1996);    -   Valentine and Owens, J. Pharmacol. Exp. Ther. 278:717-724        (1996).

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically incorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1. A compound of formula (I):

wherein: R₁ is selected from the group consisting of hydrogen andmethyl; and L is selected from the group consisting of

O(CH₂)₅ attached to the benzene ring at the meta position and

O(CH₂)₉ attached to the benzene ring at the meta position.
 2. Acomposition comprising a compound of formula (I), as claimed in claim 1,wherein L is selected from the group consisting of

O(CH₂)₅ attached to the benzene ring at the meta position, and

O(CH₂)₉ attached to the benzene ring at the meta position.
 3. Thecomposition of claim 2, further comprising an adjuvant.
 4. Thecomposition of claim 3, wherein the adjuvant is pharmaceuticallyacceptable for use in a human subject.
 5. The composition of claim 3,wherein the adjuvant is selected from the group consisting of alum,TiterMax Gold, Ribi, ASO4, Freund's complete adjuvant, and Freund'sincomplete adjuvant.
 6. The composition of claim 3, further comprising apharmaceutically acceptable carrier.
 7. The composition of claim 6,wherein the pharmaceutically acceptable carrier is a bacterial toxin ora bacterial toxoid.
 8. The composition of claim 3, further comprising apharmaceutically acceptable excipient.
 9. The compound of claim 1,wherein L is

O(CH₂)₉ attached to the benzene ring at the meta position and R¹ ismethyl.
 10. The compound of claim 1, wherein L is

O(CH₂)₅ attached to the benzene ring at the meta position and R¹ ismethyl.